Methods and products related to treatment and prevention of hepatitis c virus infection

ABSTRACT

The invention provides methods for identifying and treating subjects having hepatitis C infections. In some instances, the subjects are those that are non-responsive to non-CpG therapy. Preferably, the subjects are treated with C class CpG immunostimulatory nucleic acids having semi-soft backbone.

FIELD OF THE INVENTION

The invention provides methods and products for the treatment ofsubjects chronically infected with hepatitis C virus.

BACKGROUND OF THE INVENTION

The hepatitis C virus (HCV) is a positive strand RNA virus of theFlavivirus family that infects hepatocytes of humans and some otherprimates. First characterized in 1989 (1), HCV has a 9.5 kb genome thatencodes for three structural proteins: core and two envelopeglycoproteins (E1 and E2),' as well as several non-structural (NS)proteins that are involved in the viral replication and interaction withthe host cell (2).

HCV is a serious public health concern, causing >90% of parenteralnon-A, non-B hepatitis (1). From 0.4 to 1.5% of the world's populationis infected (3, 4), including about 300,000 Canadians (Health Canada).Epidemiological statistics are difficult to compile since the vastmajority of acute infections are subclinical; however it is estimatedthat 50-80% of HCV infected individuals fail to clear the virus, andmost of these become life-long carriers. About 50% of carriers developchronic hepatitis and 20% of these will develop liver cirrhosis, many ofwhom will subsequently develop hepatocellular carcinoma (5-9). HepatitisC causes an estimated 8,000 to 10,000 deaths annually in the UnitedStates (CDC).

In the United States and Canada, there are two different regimens, whichhave been approved as therapy for hepatitis C: monotherapy with alphainterferon and combination therapy with alpha interferon and Ribavirin.Although more expensive and associated with more side effects,combination therapy consistently yields higher rates of sustainedresponse than monotherapy.

Several forms of alpha interferon are available (alpha-2a, alpha-2b, andconsensus interferon (Alfacon)). These interferons are typically givensubcutaneously three times weekly. Pegylated interferon, i.e., alphainterferon modified by addition of polyethylene glycol (PEG) in order toincrease the duration in the circulation, is another of interferon, andit is given only once weekly. Ribavirin, in contrast, is an oralantiviral agent that is given twice a day in 200 mg capsules.

Side effects of alpha interferon include: fatigue, muscle aches,headaches, nausea and vomiting, skin irritation at the injection site,low-grade fever, weight loss, irritability, depression, suicide, mildbone marrow suppression and hair loss (reversible). For Ribavirin theside effects include; anemia fatigue and irritability, itching, skinrash, nasal stuffiness, sinusitis and cough.

Treatment with interferon alone or in combination with interferon andRibavirin leads to rapid improvements in serum ALT levels in 50-75% ofpatients and the disappearance of detectable HCV RNA from the serum in30-50% of patients. Long-term improvement in liver disease usuallyoccurs only if HCV RNA disappears during therapy and stays undetectablefor at least 6 months after therapy is completed. Combination treatmentresults in both a higher rate of loss of HCV RNA on treatment and alower rate of relapse when treatment is complete. However, resultsdepend strongly on the genotype of virus, with better results beingobtained for genotypes 2 and 3 (about 90% with 1 year of treatment withpegylated IFN-α and Ribavirin), but much poorer results (about 40%sustained response) for genotype 1 HCV. The majority of HCV chroniccarriers in North America now are of genotype 1.

The optimal duration of treatment varies depending on whether interferonmonotherapy or combination therapy is used, as well as by HCV genotype.Typically, the duration ranges from 6 to 12 months.

There is currently no vaccine against HCV, or highly effective therapyfor chronic infection. Thus there is an urgent need for an effectivetreatment that could be used to treat chronic carriers.

SUMMARY OF THE INVENTION

The invention is premised in part on several surprising findingsincluding the observation that CpG immunostimulatory nucleic acids canbe used to treat subjects that are chronically infected with hepatitis Cvirus (HCV) and that are non-responsive to previously administerednon-CpG therapies. The invention is further premised in part on theobservation that a synergistic response can be had in such subjects fromthe combined use of CpG immunostimulatory nucleic acids and ananti-viral agent such as IFN-alpha.

In one aspect, the invention provides a method of treating a subjecthaving an HCV infection that was not successfully treated using aprevious non-CpG therapy comprising administering to a subject in needof such treatment a CpG immunostimulatory nucleic acid in an amounteffective to treat the infection.

In one embodiment, the non-CpG therapy includes interferon-alpha. In arelated embodiment, the interferon-alpha is interferon-alpha-2b,interferon-alpha-2a or consensus interferon-alpha. In anotherembodiment, the non-CpG therapy includes interferon-alpha and Ribavirin,or interferon-alpha and Ribavirin and emantidine. In some importantembodiments, the non-CpG therapy includes pegylated interferon-alpha andan anti-viral such as Ribavirin.

In one embodiment, the CpG immunostimulatory nucleic acid is an A classCpG immunostimulatory nucleic acid. In another embodiment, the CpGimmunostimulatory nucleic acid is a B class CpG immunostimulatorynucleic acid.

In yet a further embodiment, the CpG immunostimulatory nucleic acid is aC class CpG immunostimulatory nucleic acid.

The method may optionally comprise administration of an anti-viral suchas interferon-alpha to the subject along with the CpG immunostimulatorynucleic acid.

The interferon-alpha may be interferon-alpha-2b, interferon-alpha-2a orconsensus interferon alpha, but is not so limited. In one embodiment,the anti-viral is administered substantially simultaneously with the CpGimmunostimulatory nucleic acid.

In one embodiment, the CpG immunostimulatory nucleic acid comprises abackbone modification. In a related embodiment, the backbonemodification is a phosphorothioate backbone modification. In someimportant embodiment, the CpG immunostimulatory nucleic acid comprises asemi-soft backbone. In other important embodiments, the CpGimmunostimulatory nucleic acid is a C class immunostimulatory nucleicacid having a semi-soft backbone.

Thus, in another aspect, a method is provided for treating a subjecthaving an HCV infection that was not successfully treated using aprevious non-CpG therapy comprising administering to a subject in needof such treatment a C class CpG immunostimulatory nucleic acid having asemi-soft backbone in an amount effective to treat the infection.

In yet another aspect, a method is provided for treating a subjecthaving an HCV infection that was not successfully treated using aprevious non-CpG therapy comprising contacting peripheral bloodmononuclear cells from a subject in need of such treatment, with a CpGimmunostimulatory nucleic acid in an amount effective to stimulate animmune response, and re-infusing the cells into the subject.

In one embodiment, the peripheral blood mononuclear cells comprisedendritic cells. In another embodiment, the dendritic cells compriseplasmacytoid dendritic cells. In one embodiment, the CpGimmunostimulatory nucleic acid is a C class immunostimulatory nucleicacid. In a related embodiment, the C class immunostimulatory nucleicacid has a semi-soft backbone.

In another aspect, the invention provides a method of treating a subjecthaving an HCV infection and likely to be non-responsive to a non-CpGtherapy comprising administering to a subject in need of such treatmenta CpG immunostimulatory nucleic acid in an amount effective to treat theinfection.

In one embodiment, the method further comprises identifying a subjectlikely to be non-responsive to a non-CpG therapy. In one embodiment, thesubject is identified as likely to be non-responsive based on an assayof interferon-alpha produced per dendritic cell. In another embodiment,the subject is identified as likely to be non-responsive based on HCVgenotype.

In one embodiment, the non-CpG therapy includes IFN-alpha. In a relatedembodiment, the non-CpG therapy includes interferon-alpha and Ribavirin.

In one embodiment, the method further comprises administering to thesubject an anti-viral agent. In important embodiments, the anti-viralagent is interferon-alpha. The interferon-alpha may beinterferon-alpha-2b, interferon-alpha-2a or consensus interferon alpha,but it is not so limited. In one embodiment, the interferon-alpha isadministered in a sub-therapeutic amount, and optionally the combinationof the CpG immunostimulatory nucleic acid and the interferon-alpha issynergistic.

In one embodiment, the CpG immunostimulatory nucleic acid used to treatthe subject is an A class CpG immunostimulatory nucleic acid, a B classCpG immunostimulatory nucleic acid, or a C class CpG immunostimulatorynucleic acid.

In one embodiment, the CpG immunostimulatory nucleic acid used toidentify whether a subject is likely to be non-responsive to a non-CpGtherapy is an A class CpG immunostimulatory nucleic acid, or a C classCpG immunostimulatory nucleic acid.

In one embodiment, the anti-viral agent is administered to the subjectsubstantially simultaneously with the CpG immunostimulatory nucleicacid. In other embodiments, the interferon-alpha is administered for aperiod prior to treatment with the CpG immunostimulatory nucleic acid.

In certain embodiments, the CpG immunostimulatory nucleic acid comprisesa backbone modification. In related embodiments, the backbonemodification is a phosphorothioate backbone modification. In somepreferred embodiments, the CpG immunostimulatory nucleic acid comprisesa semi-soft backbone, and some even more preferred embodiments, the CpGimmunostimulatory nucleic acid is a C class CpG immunostimulatorynucleic acid having a semi-soft backbone.

In another aspect, a method is provided for treating a subject having anHCV infection and likely to be non-responsive to a non-CpG therapycomprising administering to a subject in need of such treatment a Cclass CpG immunostimulatory nucleic acid having a semi-soft backbone inan amount effective to treat the infection.

In yet another aspect, the invention provides a method for screening CpGimmunostimulatory nucleic acids useful in the treatment of chronichepatitis C viral infection. The method involves contacting peripheralblood mononuclear cells from a subject having a chronic hepatitis Cviral infection, to a CpG immunostimulatory nucleic acid, and measuringa test response of the blood mononuclear cells after exposure. Thesubject from which the peripheral blood mononuclear cells wherein thesubject was not successfully treated using a previous therapy.

In one embodiment, the test response is selected from the groupconsisting of B cell stimulation, secretion of IL-6, secretion of IL-10,secretion of IL-12, secretion of interferon-gamma, secretion of type 1interferons (alpha+beta), secretion of IP-10, NK activity, expression ofCD80, expression of CD 86, expression of CD83, and upregulation of classII MHC expression.

In another embodiment, the peripheral blood mononuclear cells comprisedendritic cells. In a related embodiment, the dendritic cells compriseplasmacytoid dendritic cells. In still another embodiment, the cells aredendritic cells and the test response is selected from the groupconsisting of secretion of IL-12, secretion of type 1 interferons,expression of CD80, expression of CD 86, expression of CD83, andupregulation of class II MHC expression.

In one embodiment, the contacting occurs in vitro. In anotherembodiment, the peripheral blood mononuclear cells are cultured. In yetanother embodiment, the CpG immunostimulatory nucleic acid is added tothe cultured peripheral blood mononuclear cells.

In one embodiment, the previous therapy is a non-CpG therapy. In anotherembodiment, the non-CpG therapy comprises interferon-alpha. In anotherembodiment, the non-CpG therapy further comprises Ribavirin. In otherembodiments, the interferon-alpha is pegylated interferon-alpha. In oneembodiment, the previous therapy is therapy with a CpG nucleic acid of adifferent sequence or class.

In other embodiments, the method further comprises screening the CpGimmunostimulatory nucleic acid for the ability to stimulate a controlresponse from peripheral blood mononuclear cells from a normal subject.

The method may further comprise contacting peripheral blood mononuclearcells to interferon-alpha substantially simultaneously with the CpGimmunostimulatory nucleic acid.

In one embodiment, the CpG immunostimulatory nucleic acid comprises abackbone modification. In a related embodiment, the backbonemodification is a phosphorothioate backbone modification. In importantembodiments, the CpG immunostimulatory nucleic acid comprises asemi-soft backbone. The CpG immunostimulatory nucleic acid may be an Aclass CpG immunostimulatory nucleic acid, a B class CpGimmunostimulatory nucleic acid, or a C class CpG immunostimulatorynucleic acid. In some embodiments, the CpG immunostimulatory nucleicacid is a C class immunostimulatory nucleic acid, and in otherembodiments, the CpG immunostimulatory nucleic acid is a C classimmunostimulatory nucleic acid with a semi-soft backbone.

In another aspect, the invention provides a method for identifying asubject having an HCV infection and likely to be non-responsive to anon-CpG therapy. The method involves exposing peripheral bloodmononuclear cells harvested from a subject having a hepatitis C viralinfection to a CpG immunostimulatory nucleic acid, measuringinterferon-alpha produced from the cells, and determining an amount ofinterferon-alpha produced per dendritic cell, wherein an amount that isbelow 1.0 pg/ml is indicative of a subject that is likely to benon-responsive to a non-CpG therapy. In one embodiment, an amount thatis below 0.5 pg/ml is indicative of a subject that is likely to benon-responsive to a non-CpG therapy.

In one embodiment, the non-CpG therapy comprises interferon-alpha. Inanother embodiment, the non-CpG therapy comprises Ribavirin. In anotherembodiment, the IFN-alpha is pegylated IFN-alpha.

In some important embodiments, the CpG immunostimulatory nucleic acid isan A class or a C class CpG immunostimulatory nucleic acid.

In still other embodiments, the peripheral blood mononuclear cells arefurther exposed to an anti-viral agent together with a CpGimmunostimulatory nucleic acid. The anti-viral agent may beinterferon-alpha, but it is not so limited. In one embodiment, theinterferon-alpha is interferon-alpha-2b, interferon-alpha-2a orconsensus interferon alpha.

In one embodiment, the peripheral blood mononuclear cells comprisedendritic cells. In another embodiment, the dendritic cells compriseplasmacytoid dendritic cells.

In another embodiment, the hepatitis C viral infection is an acutehepatitis C viral infection.

In another embodiment, the method further comprises determining agenotype of the HCV.

In still a further aspect, a method is provided for identifying asubject having an HCV infection and likely to be non-responsive to anon-CpG therapy comprising exposing peripheral blood mononuclear cellsharvested from a subject having a hepatitis C viral infection to an Aclass or a C class CpG immunostimulatory nucleic acid, measuringinterferon-alpha produced from the cells, and determining an amount ofinterferon-alpha produced per dendritic cell, wherein an amount that isbelow 1.0 pg/ml is indicative of a subject that is likely to benon-responsive to a non-CpG therapy.

In yet another aspect, the invention provides a method of treating asubject having a hepatitis C viral infection comprising administering toa subject, identified according to the method described above, a CpGimmunostimulatory nucleic acid molecule in an amount effective to treatthe infection.

In one embodiment, the method further comprises administering to thesubject interferon-alpha. In one embodiment, the interferon-alpha isinterferon-alpha-2b, interferon-alpha-2a or consensus interferon-alpha.

In one embodiment, the CpG immunostimulatory nucleic acid used to treatthe subject is an A class CpG immunostimulatory nucleic acid, a B classCpG immunostimulatory nucleic acid, or a C class CpG immunostimulatorynucleic acid.

In another embodiment, the CpG immunostimulatory nucleic acid comprisesa backbone modification. In a related embodiment, the backbonemodification is a phosphorothioate backbone modification. In yet anotherembodiment, the CpG immunostimulatory nucleic acid comprises a semi-softbackbone.

In one embodiment, the hepatitis C viral infection is a chronichepatitis C viral infection. In another embodiment, the hepatitis Cviral infection is an acute hepatitis C viral infection.

Each of the limitations of the invention can encompass variousembodiments, of the invention. It is, therefore, anticipated that eachof the limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

These and other aspects of the invention are described in greater detailbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the induction of IFN-α secretion from HCV-infected andnormal PBMCs following stimulation with 3 classes of CpG. PBMCs fromnormal or HCV-infected subjects were incubated with different classes ofCpG for 48 h. Cell supernatants were collected and assayed for IFN-αsecretion by commercial ELISA kits. The average IFN-α secretion for 10normal subjects and 10 HCV-infected subjects are shown by the blackbars.

FIG. 2 shows the flow cytometric analysis of freshly isolated PBMCs fromchronic HCV carriers and normal subjects. PBMCs were isolated from theblood of HCV infected subjects and from normal healthy donors andimmunostained with fluorescent-tagged anti-plasmacytoid dendritic cell(pDC) antibodies. Cells were analyzed on a flow cytometer and resultswere compared to IFN-α secretion data on these same subjects whenstimulated with CpG.

FIG. 3 shows the IFN-α induction by stimulation of PBMCs with C-classand soft-C oligonucleotides. PBMCs from normal or HCV-infected subjectswere incubated with different classes of CpG for 48 h. Cell supernatantswere collected and assayed for IFN-α secretion by commercial ELISA kits.The average IFN-α secretion for 10 normal subjects and 10 HCV-infectedsubjects are shown by the black bars.

FIG. 4 shows the IFN-α induction following stimulation with a panel ofsemi-soft C-class CpG. PBMCs isolated from 5 HCV-infected subjects wereincubated with a panel of semi-soft C-class oligonucleotides for 48 h.Cell supernatants were collected and assayed for ITN-α secretion bycommercial ELISA kits. The average IFN-α secretion for 5 HCV-infectedsubjects are shown by the black bars.

FIG. 5 shows the IFN-γ secretion following stimulation with threeclasses of CpG. PBMCs from normal or HCV-infected subjects wereincubated with different classes of CpG for 48 h. Cell supernatants werecollected and assayed for IFN-γ secretion by commercial ELISA kits. Theaverage IFN-γ secretion for 10 normal subjects and 10 HCV-infectedsubjects are shown by the black bars.

FIG. 6 shows the IFN-γ induction following stimulation with a panel ofsemi-soft C-class CpG. PBMCs isolated from 5 HCV-infected subjects wereincubated with a panel of semi-soft C-class oligonucleotides for 48 h.Cell supernatants were collected and assayed for IFN-γ secretion bycommercial ELISA kits. The average IFN-γ secretion for 5 HCV subjectsare shown by the black bars.

FIG. 7 shows the IP-10 secretion following stimulation with threeclasses of CpG. PBMCs from normal or HCV-infected subjects wereincubated with different classes of CpG for 48 h. Cell supernatants werecollected and assayed for IP-10 secretion by commercial ELISA kits. Theaverage IP-10 secretion for 10 normal subjects and 10 HCV-infectedsubjects are shown by the black bars.

FIG. 8 shows the effect of CpG on B cell proliferation. PBMCs fromHCV-infected or normal donors were incubated with class A, B or C CpGfor 5 days. Cells were then pulsed with ³H-thymidine for 16 to18 hoursbefore measuring radioactivity. Values are represented as stimulationindices in comparison with media control (SI=cpm incubated with CpG/cpmof cells incubated with media alone).

FIG. 9 shows the effect of semi-soft C-class CpG on B cellproliferation. PBMCs from 5 HCV-infected subjects were incubated with A,B,C and semi-soft-C class CpG for 5 days. Cells were then pulsed with³H-thymidine for 16 to 18 hours before measuring radioactivity. Valuesare represented as stimulation indices in comparison with media control(SI=cpm incubated with CpG/cpm of cells incubated with media alone).

FIG. 10 shows the IL-10 secretion following stimulation with threeclasses of CpG. PBMCs from normal or HCV-infected subjects wereincubated with different classes of CpG for 48 h. Cell supernatants werecollected and assayed for IL-10 secretion by commercial ELISA kits. Theaverage IL-10 secretion for 10 normal subjects and 10 HCV-infectedsubjects are shown by the black bars.

FIG. 11 shows IFN-α secretion following stimulation of HCV-infectedcells with Ribavirin and CpG alone or in combination with Intron A.PBMCs from 10 HCV-infected subjects and 10 normal healthy donors wereincubated with Intron A, Ribavirin or C-class CpG alone and also withand without Intron A (a purified exogenous source of IFN-α) for 48hours. Cell supernatants were collected and assayed for IFN-α secretionby commercial ELISA kits. The amount of IFN-α measured for Intron Aalone for each subject, was considered background and was subtractedfrom Intron A, Ribavirin+Intron A and C-Class+Intron A for these samesubjects before the data was included in the graph. Mean values fornormal and HCV subjects are indicated by black and white barsrespectively.

FIG. 12. Synergistic effect of CpG combined with Intron A on IFN-αsecretion by HCV-infected cells. PBMCs from 15 HCV-infected subjectswere incubated with C-class CpG alone or together with Intron A (apurified exogenous source of IFN-α) for 48 hours. Cell supernatants werecollected and assayed for IFN-α secretion by commercial ELISA kits. Theamount of IFN-α measured for Intron A alone for each subject, wassubtracted from CpG+Intron A for these same subjects before the data wasincluded in the graph.

It is to be understood that the figures are not required for enablementof the claimed invention.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered according to the invention, that CpGoligonucleotides can activate PBMCs from patients chronically infectedwith HCV, including those who have failed previous interferon-alpha(IFN-α) therapy, in a manner similar to PBMCs from healthy subjects.

It was discovered that endogenous IFN-α secretion was strongly inducedfrom plasmacytoid dendritic cells (PDC), which are thought to beinfected by HCV resulting in their dysfunction and reduced ability torespond to other stimuli. In some instances, the A and C CpG classes,which induce high levels of IFN-α in PBMCs from healthy volunteers, werefound to induce the highest levels of IFN-α from pDCs. It was furtherdiscovered that the semi-soft C class CpG ODN are also particularlyuseful for this effect. These ODN may be preferred in some embodimentssince they will not accumulate in the kidney with repeat dosing.

It has been further discovered according to the invention that neitherexogenous IFN-α (Intron A) nor Ribavirin have any detectable directimmune stimulatory effects on PBMCs from normal subjects or HCV chroniccarriers, when used alone or together. However, when Intron A and CpGODN (e.g., B or C classes) are used together, then a strong synergy forproduction of endogenous IFN-α is observed.

These results indicate that CpG ODN are an effective treatment alone, ortogether with IFN-α, to treat chronic HCV infection. The inventionprovides methods and products for preventing and treating HCV infection,based on these findings.

Chronic infection appears to be due, at least in part, to the rapidmutation rate of HCV, resulting in the production of quasi-species thatcan escape immune surveillance (10, 11). Both humoral and cell-mediatedimmune (CMI) responses can be detected in chronically infectedindividuals. While neutralizing antibodies are critical to protectionfrom infection, cell-mediated immunity (CMI) appears to play the majorrole in viral clearance once infection is established.

In one aspect, the invention provides a method of treating a subjectinfected with hepatitis C virus (HCV) who is not successfully treatedwith a previous non-CpG therapy. The method comprises administering to anon-responsive subject in need of such treatment a CpG immunostimulatorynucleic acid in an amount effective to inhibit the infection.

A non-CpG therapy, as used herein, is a therapy that uses active orinactive compounds that are not CpG immunostimulatory nucleic acids. Invarious embodiments, the non-CpG therapy includes interferon-alpha.Pegylated IFN-alpha is commonly administered to HCV subjects (e.g.,human HCV patients), preferably in combination with Ribavirin andoptionally amantadine. The interferon-alpha can be interferon-alpha-2b,interferon-alpha-2a or consensus interferon-alpha. All of the foregoinginterferon-alpha treatments are included in the definition of non-CpGtherapy.

A subject who is not successfully treated with a previous non-CpGtherapy is a subject who notwithstanding prior treatment still hasdetectable viral load in their bloodstream 6 months after the cessationof therapy. These subjects include those that may respond to a previousnon-CpG therapy, but who fail to control the infection and subsequentlyrelapse as indicated by detectable viral load. As used herein and forthe sake of simplicity, these subjects are referred to as“non-responders”, however this term is to be understood as definedherein, and not as defined in a clinical setting. In other words,although in a clinical setting a “non-responder” defines only thatnarrow subset of subjects that fail to show any response to a treatment,the invention is directed to a broader category of subjects that whileperhaps responding at some level to a previous treatment, are still notsuccessfully treated. A subject that is successfully treated is one thathas no detectable viral load in its bloodstream 6 months after thecessation of treatment. Successful treatment means treatment that leadsto an undetectable level of viral load in the bloodstream that issustained for at least 6 months after cessation of treatment. It is tobe understood that a non-responder, as used herein, implicitly is alsochronically infected with HCV.

As used herein, when referring to treatment using CpG nucleic acids, themethods are used to achieve a successful treatment of subjects.Successful treatment of subjects using CpG treatment is defined as areduction of viral load to undetectable levels in the bloodstream 6months after the cessation of therapy. Interestingly, viral loads maynot be observed to decrease during or immediately after CpG treatment,but rather may only decrease with time after treatment, with theultimate result that there is no detectable virus in the bloodstream ofthese subjects 6 months following the cessation of treatment. To treatan infection therefore means to reduce viral load to an undetectablelevel in the bloodstream of a subject and to sustain that level for 6months following the cessation of treatment. Effective amounts of agentsare therefore administered to achieve this end result.

In some embodiments, the potential non-responders may be identifiedprospectively (i.e., prior to actual in vivo treatment with a non-CpGtherapy), and the invention provides methods not only for theidentification of such subjects but also for their treatment. Potentialnon-responders may be identified by assessing their ability to respondto CpG immunostimulatory nucleic acids, particularly A class and Cclass. The ability to respond to CpG immunostimulatory nucleic acidswill be assessed by the amount of interferon-alpha that is produced perpDC in HCV infected subjects. It was discovered, according to theinvention, that HCV infected subjects that would be unlikely to respondto non-CpG therapy such as IFN-alpha therapy could be identified priorto receiving such treatment. The ability to identify such subjects priorto in vivo therapy eliminates unnecessary treatment and places thesubjects in a therapeutically advantageous position for treatment withthe CpG immunostimulatory nucleic acids of the invention either alone orin combination with other anti-HCV therapies including but not limitedto IFN-alpha. These subjects would suffer from less cytotoxicity and thetime period for viral growth would be reduced by not undergoing atreatment that will be unsuccessful. Subjects having below a reasonablelevel of IFN-alpha induction per pDC are likely not to be successfullytreated with IFN-alpha and thus should be treated using the methodsprovided herein. Measurement of IFN-alpha induction and pDC numbers aredescribed in more detail in the Examples.

It is to be understood that an HCV infected subject that is successfullytreated with any of the therapeutic agents and methods discussed hereinwill probably still have virus in their body. However, while the subjectis not able to completely eradicate the virus, it is able to controlviral load (to undetectable levels). Although not intending to be boundby any particular theory, it is expected that the maintenance ofundetectable viral loads in such subjects involve an immune system thatis able to control viral replication and spread.

One of ordinary skill given the teachings provided herein will be ableto determine whether a subject is likely to be a ‘non-responder” toIFN-alpha therapy. As an example, if the IFN-alpha induction wereperformed with an A class nucleic acid such as nucleic acid designatedSEQ ID NO 1, under the culture conditions described in the Examples,then a normal response indicative of the ability to respond to IFN-alphatherapy would be at least 1 pg/ml per pDC. An amount less than this isindicative of some pDC dysfunction. Amounts that are less than 0.5 pg/mlper pDC correlate with a higher probability of non-response to IFN-alphatreatment. One of ordinary skill will be able to determine such cutoffsfor the particular type of nucleic acid used in the assay, and willtherefore be capable of identifying subjects expected not to besuccessfully treated with MN-alpha therapy (at least) prior to actuallytreating such subjects in that manner.

In still other embodiments, the method of identifying a subject who islikely to be a non-responder to non-CpG therapy (e.g., IFN-alphatherapy) may further include identification of the genotype of HCVhe/she is infected with. It is more likely that a subject infected witha genotype 1 HCV will not be successfully treated with IFN-alphatherapy, for example. Therefore, in addition to assessing the productionof IFN-alpha per DC in such subjects, their HCV genotype can also bedetermined (using methods known in the art), and this combination ofinformation can be used to identify a subject that is likely to benon-responsive to IFN-alpha therapy.

It is to be further understood that in some aspects, the inventionprovides a method for identifying a subject that is unlikely to besuccessfully treated using a non-CpG therapy (without actually treatingthe subject with a non-CpG therapy) and then treating the subject usingeither CpG immunostimulatory nucleic acids alone or in combination withan anti-viral agent such as but not limited to IFN-alpha.

The above methods can also be used to screen subjects for their responseto particular CpG immunostimulatory nucleic acids.

In still other embodiments, the methods may involve the additional stepof identifying subjects having received previous non-CpG therapy but notsuccessfully treated. Those of ordinary skill, given the teachingsprovided herein, will be able to identify such subjects. As an example,such subjects would have detectable viral loads in their bloodstream 6months after the cessation of treatment. In some embodiments, thesesubjects may also demonstrate a reduction in viral load immediatelyfollowing treatment, but this reduction is not sustained.

The invention intends to treat subjects not successfully treated with aprevious non-CpG therapy using, inter alia, CpG immunostimulatorynucleic acids alone or in combination with other active agents such asthose previously described for HCV infection. As broadly defined, CpGimmunostimulatory nucleic acids are nucleic acids having at least oneCpG dinucleotide motif in which at least the C of the dinucleotide isunmethylated. CpG immunostimulatory nucleic acids include but are notlimited to A class, B class and C class CpG immunostimulatory nucleicacids, as described more fully herein and in the patent and patentapplications cited herein and incorporated by reference. These classesof CpG immunostimulatory nucleic acid have differing properties andactivation profiles.

In important embodiments, the CpG immunostimulatory nucleic acid is a Cclass immunostimulatory nucleic acid. It was surprisingly found,according to the invention, that C class immunostimulatory nucleic acidswere preferred in some embodiments, even though these nucleic acidspossessed properties intermediate to those of A class and B class. TheExamples provided herein demonstrate that, even though pDC ofchronically infected subjects not successfully treated with a previousnon-CpG therapy are themselves infected with HCV and therebydysfunctional in some aspects, exposure of such cells to CpGimmunostimulatory nucleic acids, and in particular C classimmunostimulatory nucleic acids, restores their function. In someembodiments, it is also preferred that the C class immunostimulatorynucleic acids be either of a “soft” or “semi-soft” variety, as describedin greater detail herein. In some preferred embodiments, the CpGimmunostimulatory is a semi-soft C class nucleic acid.

In other aspects, the CpG immunostimulatory nucleic acids are used incombination with active agents which preferably include those previouslydescribed for HCV treatment. Of particular importance is the use of CpGimmunostimulatory nucleic acids with interferon-a (e.g., Intron A). Theinterferons that can be used in combination with the CpGimmunostimulatory nucleic acids of the invention include but are notlimited to interferon-alpha-2b, interferon-alpha-2a or consensusinterferon alpha. Other anti-virals are described herein. Any of the CpGclasses can be used in these combinations. As an example, it wasunexpectedly found, according to the invention, that althoughexogenously administered interferon-a fails to treat these subjectssuccessfully, when combined with CpG immunostimulatory nucleic acids itis therapeutically efficacious. In some embodiments, the CpGimmunostimulatory nucleic acid is a C class immunostimulatory nucleicacid. In come preferred embodiments, it is a semi-soft C class nucleicacid.

The timing of administration of the CpG nucleic acid and anti-viralagent (e.g., interferon-alpha) may vary depending upon the subject andthe severity of infection. The CpG nucleic acid may be administeredsubstantially simultaneously with the CpG immunostimulatory nucleicacid. This means that the two agents may be combined prior toadministration, or may be combined in the process of administration(e.g., with both feeding into an intravenous line in a subject), or theymay be administered separately but within a period of time that it wouldtake someone to perform two administrations (e.g., the time to inject asubject twice). Regardless of whether the agents are administeredsubstantially simultaneously or in staggered fashion, the order mayvary. Accordingly, in some embodiments, the CpG immunostimulatorynucleic acid may be administered prior to an anti-viral agent such asIFN-alpha while in others it may be administered following theanti-viral agent.

When CpG nucleic acids are used together with other anti-virals (e.g.,IFN-alpha), these compounds may be administered in a combined amountthat is therapeutically efficacious. The amount of either compound maytherefore be sub-therapeutic or supra-therapeutic (i.e., below or abovethe amount that would be therapeutically efficacious when administeredalone). Alternatively, the compounds each may be administered in atherapeutic amount, but the combination of those agents creates atherapeutic benefit such as a reduction of side effects. In preferredembodiments, if the anti-viral is IFN-alpha, it is administered in atherapeutic amount. Regardless of the actual amounts administered, thecombination of agents may be synergistic. A synergistic response is onethat is greater than the additive response expected by the combinationof the agents.

In still other aspects and in keeping with the description providedabove, the invention provides methods for screening CpG nucleic acidsfor the ability to stimulate immune cells isolated from a subjectchronically infected with HCV and not successfully treated with anon-CpG therapy or likely to be non-responders to non-CpG therapy. Thesescreening methods are generally performed in vitro by contactingperipheral blood mononuclear cells (PBMCs) with a CpG immunostimulatorynucleic acid in an effective amount sufficient to stimulate an immuneresponse. The immune response can be measured by any number of markers,including IFN-alpha production, B cell stimulation, secretion ofcytokines such as 11-6, IL-10, IL-12, interferon-gamma, type 1interferons (alpha+beta), chemokine secretion such as IP-10, NKactivity, expression of costimulatory molecules (e.g., CD80, CD 86) andmaturation molecules (e.g., CD83) and upregulation of class II MHCexpression.

In some important embodiments, the immune cells are dendritic cells, andpreferably plasmacytoid dendritic cells (pDCs) and the immune responsemarkers are specific to this cell type. These include but are notlimited to expression of costimulatory molecules (e.g., CD80 and CD86)expression of maturation molecules (e.g., CD83), expression and/orsecretion of IL-12 and type 1 interferons (alpha+beta), and upregulationof class II MHC expression. It is to be understood that these in vitroassays are not dependent upon isolation of dendritic cells such as pDCsfrom the remainder of PBMCs. Rather the assays can be carried out inhomogeneous populations of PBMCs.

In still another aspect, the invention provides a method for identifyinga subject having a chronic hepatitis C viral infection to be treatedwith a CpG immunostimulatory nucleic acid. The method involves exposingperipheral blood mononuclear cells harvested from a subject having achronic hepatitis C viral infection to i) a CpG immunostimulatorynucleic acid, and ii) a CpG immunostimulatory nucleic acid and ananti-viral (e.g., interferon-alpha), and measuring response of theperipheral blood mononuclear cells after exposure. A response to a CpGimmunostimulatory nucleic acid is indicative of a subject to be treatedwith a CpG immunostimulatory nucleic acid either following or in placeof a non-CpG therapy (as described above, but only after identifying asubject that is unlikely to respond to a non-CpG therapy). A response toa CpG immunostimulatory nucleic acid together with an anti-viral agent(e.g., interferon-alpha) that greater than the response to CpGimmunostimulatory nucleic acid alone is indicative of a subject to betreated with the combination. As described herein, the anti-viral agentcan be an interferon-alpha including but not limited tointerferon-alpha-2b, interferon-alpha-2a or consensus interferon-alpha.Preferably, the peripheral blood mononuclear cells comprise dendriticcells such as plasmacytoid dendritic cells. The invention furtherincludes treatment of subjects identified as just described using eitherCpG immunostimulatory nucleic acids alone or in combination with ananti-viral agent (e.g., IFN-alpha), depending upon the outcome of thescreening assay. Clinical strategies comprise local and systemic in vivoadministration of such nucleic acids, as well as ex vivo strategies inwhich pDCs isolated from non-responsive HCV infected subjects areactivated in vitro with immunostimulatory nucleic acids and thenreinfused into the patient locally or systemically. These therapeuticstrategies may include the combination with other growth factors (IL-3,GM-CSF, flt3-ligand, etc.) as well as with other stimuli (superantigens,viral products). Since natural IFN-α is a family of more than a dozenseparate gene products, the individual products of which have uniqueactivity profiles, the clinical use of natural interferon may bepreferable compared to recombinant IFN-α derived from a singlerecombinant IFN-α gene.

The invention further provides a method activating pDCs from anHepatitis C infected subject. The method involves isolating pDCs fromthe subject in need of such treatment, culturing the isolated pDCs invitro, contacting the pDCs in vitro with an effective amount of anisolated immunostimulatory nucleic acid, and returning the contactedcells to the subject. The cells can also be contacted in vitro with agrowth factor or with a cytokine. The immunostimulatory nucleic acidsand conditions calling for treatment with IFN-α according to this aspectof the invention are as described above.

IFN-alpha itself represents a family of more than a dozen related,homologous proteins (isoforms, see Table 1 below), each encoded by aunique gene and each exhibiting a unique activity profile. Theactivities of the different alpha-interferon species on viruses can varyas much as twenty-fold or more. IFN-alpha products in clinical use arerecombinant proteins or highly purified natural proteins of a singleisoform. In the United States IFN-α is available as recombinant humanIFN-α2a (ROFERON-A), recombinant human IFN-α2b (INTRON A), and aspurified natural IFN-αn3 (ALFERON N). Outside the United States, IFN-αis also available as purified natural IFN-αn1 (WELLFERON).

TABLE 1 Family of Human IFN-α IFN-αA (IFN-α2a) IFN-α2 (IFN-α2b) IFN-α4b(IFN-α4) IFN-αB2 (IFN-α8) IFN-αC (IFN-α10) IFN-αD (IFN-α1) IFN-αF(IFN-α21) IFN-αG (IFN-α5) IFN-αH2 (IFN-α14) IFN-αI (IFN-α17) IFN-αJ1(IFN-α7) IFN-αK (IFN-α6) IFN-αM1 IFN-αN IFN-αWA (IFN-α16)

Some of the methods of the invention require measurement of immuneresponses including detecting the presence of IFN-α. Assays for IFN-αare well known in the art. These include direct tests, e.g.,enzyme-linked immunosorbent assay (ELISA) specific for at least oneIFN-α, and indirect tests, e.g., functional tests including NK cellactivation/cytotoxicity (Trinchieri G Adv Immunol 47:187-376 (1989) andphenotyping by fluorescence-activated cell sorting (FACS) analysis forclass I MHC. Additional specific assay methods well known in the art canbe particularly useful in settings where local concentration or localpresence of IFN-α is of interest. These methods include, for example,immunohistochemistry, nucleic acid hybridization (e.g., Northernblotting), Western blotting, reverse transcriptase/polymerase chainreaction (RT/PCR), and in situ RT/PCR. Intracellular IFN-α can also bedetected using flow cytometry.

The invention in some aspects involves measuring pDC activation. pDCactivation can be assayed in a number of ways. These include IFN-αproduction, expression of costimulatory molecules (e.g., CD80 and CD86),expression of maturation molecules (e.g., CD83), expression of IL-12,and upregulation of class II MHC expression. Unlike administration ofexogenous IFN-α, activation of pDC leads to the production of various ifnot all the forms of IFN-α, as well as other type I IFN such as IFN-β.In some embodiments, therefore, the pDC are activated as measured bytheir ability to produce type I interferons including IFN-α.

The invention provides various methods that involve immunostimulatorynucleic acids. An immunostimulatory nucleic acid is a nucleic acidmolecule which, upon contacting cells of the immune system, is itselfcapable of inducing contacted cells of the immune system to proliferateand/or to become activated. The contacting can be direct or indirect,e.g., the immunostimulatory nucleic acid may directly stimulate a firsttype of immune cell to express a product which may in turn stimulate asecond type of immune cell which has not been exposed to, or is notresponsive to, the immunostimulatory nucleic acid. The immunostimulatoryeffect of the immunostimulatory nucleic acid is separate from anyproduct that might happen to be encoded by the sequence of theimmunostimulatory nucleic acid. Similarly, the immunostimulatory effectof an immunostimulatory nucleic acid is distinct from and does not relyupon any antisense mechanism.

Only certain nucleic acids are immunostimulatory nucleic acids.Originally it was believed that certain palindromic sequences wereimmunostimulatory. Tokunaga T et al. Microbiol Immunol 36:55-66 (1992);Yamamoto T et al. Antisense Res Dev 4:119-22 (1994). Further workdemonstrated that non-palindromic sequences are also immunostimulatoryprovided they contained CpG dinucleotides within particular sequencecontexts (CpG motifs). Krieg A M et al. Nature 374:546-9 (1995).

The immunostimulatory nucleic acids can be single-stranded ordouble-stranded. Generally, double-stranded nucleic acid molecules aremore stable in vivo, while single-stranded nucleic acid molecules haveincreased immune activity. Thus in some aspects of the invention it ispreferred that the immunostimulatory nucleic acid be single-stranded andin other aspects it is preferred that the immunostimulatory nucleic acidbe double-stranded.

The methods and products provided in accordance with the inventionrelate to the use of CpG oligonucleotides. CpG ODN trigger most (>95%)B-cells to proliferate, secrete immunoglobulin (Ig), IL-6 and IL-12, andto be protected from apoptosis. In addition, CpG ODN cause DC maturationand also directly activate DCs, monocytes, and macrophages to secreteIFN-α/β, IL-6, IL-12, GM-CSF, chemokines and TNF-α. These cytokinesstimulate natural killer (NK) cells to secrete IFN-γ and have increasedlytic activity. Overall, CpG induces a strong Th1-like pattern ofcytokine production dominated by IL-12 and IFN-γ with little secretionof Th2 cytokines.

In addition to induction of innate immune responses, CpG DNA alsoaugments antigen-specific responses due to (i) a strong synergy betweenthe B-cell signaling pathways triggered through the B-cell antigenreceptor and by CpG, (ii) Th1-like cytokines that replace or augmentantigen-specific T-help augmenting both B- and T-cell antigen-specificresponses and (iii) up-regulation of co-stimulatory molecules that arerequired for cellular responses.

CpG ODN has been shown to be a potent adjuvant to HBsAg in BALB/c micewith clear Th1-like responses (predominantly IgG2a antibodies and strongCTL) (49). CpG ODN was found to be superior to other Th1 adjuvants suchas monophosphoryl lipid A (MPL, Corixa) or even complete Freund'sadjuvant (CFA) which is too toxic for human use. Similar results havebeen reported using CpG ODN with a variety of other antigens (47,50-53). CpG ODN have also been reported to redirect a Th2 responsepreviously established by immunization with a Th2 antigen (i.e.,Schistosomiasis surface antigen) (54) or a Th2 adjuvant (i.e., alum).

There are at least three basic classes of CpG ODNs found to be effectiveat stimulating healthy human PBMCs (Table 1). These have differentialeffects that are likely associated with the different modes of by whichCpG ODNs can stimulate immune cells.

The B class of CpG ODN are synthesized with nuclease resistantphosphorothioate backbones and are generally characterized by goodB-cell and DC activation, leading to the production of IL-12 andantibody, but only limited NK cell activation. This class of ODNfunctions well as a vaccine adjuvant, as has already been demonstratedin a phase FIT clinical trial testing CpG (a member of this class) (SEQID NO.: 2) as an adjuvant to a commercial hepatitis B vaccine (60).

The A class of CpG ODNs are synthesized with a chimeric backbone wherethe 5′ and 3′ ends are phosphorothioate and the central CpG motif regionis phosphodiester. These ODNs are characterized by good NK cell and DCactivation leading to greater production of IFN-α but limited B-cellactivation.

The C class of CpG ODN are synthesized with a phosphorothioate backboneand have stimulatory properties intermediate to the other two classes ofCpG ODNs (e.g., good activation of B-cells as well as activation of NKcells and DCs).

TABLE 1 Pattern of in vitro immune activation induced by the threedifferent classes of CpG ODNs Natural Class Backbone B-cells Killercells Dendritic cells IFN-α A SOS² + ++++ ++++ ++++ B S¹ ++++ ++ ++++ +C S¹ +++ +++ +++ +++ ¹S-ODN are made with a phosphorothioate backbone²SOS-ODN are made with a chimeric backbone where the centralCpG-containing region has phosphodiester linkages and the 3′ and 5′ endsof the ODN are made with phosphorothioate linkages

The methods of the invention may embrace the use of A class, B class andC class CpG immunostimulatory nucleic acids. As to CpG nucleic acids, ithas recently been described that there are different classes of CpGnucleic acids. One class is potent for activating B cells but isrelatively weak in inducing IFN-α and NK cell activation; this class hasbeen termed the B class. The B class CpG nucleic acids typically arefully stabilized and include an unmethylated CpG dinucleotide withincertain preferred base contexts. See, e.g., U.S. Pat. Nos. 6,194,388;6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another classis potent for inducing IFN-α and NK cell activation but is relativelyweak at stimulating B cells; this class has been termed the A class. TheA class CpG nucleic acids typically have stabilized poly-G sequences at5′ and 3′ ends and a palindromic phosphodiester CpGdinucleotide-containing sequence of at least 6 nucleotides. See, forexample, published patent application PCT/US00/26527 (WO 01/22990). Yetanother class of CpG nucleic acids activates B cells and NK cells andinduces IFN-α; this class has been termed the C-class. The C-class CpGnucleic acids, as first characterized, typically are fully stabilized,include a B class-type sequence and a GC-rich palindrome ornear-palindrome. This class has been described in U.S. provisionalpatent application 60/313,273, filed Aug. 17, 2001, U.S. Ser. No.10/224,523 filed on Aug. 19, 2002, and US the entire contents of whichare incorporated herein by reference.

“A class” CpG immunostimulatory nucleic acids have been described inU.S. Non-Provisional Patent Application Serial No.: 09/672,126 andpublished PCT application PCT/US00/26527 (WO 01/22990), both filed onSep. 27, 2000. These nucleic acids are characterized by the ability toinduce high levels of interferon-alpha while having minimal effects on Bcell activation. The A class CpG immunostimulatory nucleic acid do notnecessarily contain a hexamer palindrome GACGTC, AGCGCT, or AACGTTdescribed by Yamamoto and colleagues. Yamamoto S et al. J. Immunol148:4072-6 (1992).

Exemplary sequences of A class immunostimulatory nucleic acids aredescribed in U.S. Non-Provisional patent application Ser. No.:09/672,126 and published PCT application PCT/US00/26527 (WO 01/22990),both filed on Sep. 27, 2000.

B class CpG immunostimulatory nucleic acids strongly activate human Bcells but have minimal effects inducing interferon-a. B class CpGimmunostimulatory nucleic acids have been described in U.S. Pat. Nos.6,194,388 B1 and 6,239,116 B1, issued on Feb. 27, 2001 and May 29, 2001respectively.

The CpG oligonucleotides of the invention are oligonucleotides whichinclude at least one unmethylated CpG dinucleotide. An oligonucleotidecontaining at least one umnethylated CpG dinucleotide is a nucleic acidmolecule which contains an unmethylated cytosine-guanine dinucleotidesequence (i.e., “CpG DNA” or DNA containing a 5′ cytosine followed by 3′guanine and linked by a phosphate bond) and activates the immune system.The entire CpG oligonucleotide can be unmethylated or portions may beunmethylated but at least the C of the 5′ CG 3′ must be umnethylated.The terms CpG oligonucleotide or CpG nucleic acid as used herein referto an immunostimulatory CpG oligonucleotide or a nucleic acid unlessotherwise indicated.

In one embodiment the invention provides a B class CpG oligonucleotiderepresented by at least the formula:

5′ X₁X₂CGX₃X₄ 3′

wherein X₁, X₂, X₃, and X₄ are nucleotides. In one embodiment X₂ isadenine, guanine, or thymine. In another embodiment X₃ is cytosine,adenine, or thymine

In another embodiment the invention provides an isolated B class CpGoligonucleotide represented by at least the formula:

5′ N₁X₁X₂CGX₃X₄N₂ 3′

wherein X₁, X₂, X₃, and X₄ are nucleotides and N is any nucleotide andN₁ and N₂ are nucleic acid sequences composed of from about 0-25 N'seach. In one embodiment X₁X₂ is a dinucleotide selected from the groupconsisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT,and TpG; and X₃X₄ is a dinucleotide selected from the group consistingof: TpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.Preferably X₁X₂ is GpA or GpT and X₃X₄ is TpT. In other embodiments X₁or X₂ or both are purines and X₃ or X₄ or both are pyrimidines or X₁X₂is GpA and X₃ or X₄ or both are pyrimidines. In another preferredembodiment X₁X₂ is a dinucleotide selected from the group consisting of:TpA, ApA, ApC, ApG, and GpG. In yet another embodiment X₃X₄ is adinucleotide selected from the group consisting of: TpT, TpA, TpG, ApA,ApG, GpA, and CpA. X₁X₂ in another embodiment is a dinucleotide selectedfrom the group consisting of: TpT, TpG, ApT, GpC, CpC, CpT, TpC, GpT andCpG; X₃ is a nucleotide selected from the group consisting of A and Tand X₄ is a nucleotide, but wherein when X₁X₂ is TpC, GpT, or CpG, X₃X₄is not TpC, ApT or ApC.

In another preferred embodiment the CpG oligonucleotide has the sequence5′ TCN₁TX₁X₂CGX₃X₄ 3′ (SEQ ID NO.:26). The CpG oligonucleotides of theinvention in some embodiments include X₁X₂ selected from the groupconsisting of GpT, GpG, GpA and ApA and X₃X₄ is selected from the groupconsisting of TpT, CpT and TpC.

The B class CpG nucleic acid sequences of the invention are thosebroadly described above as well as disclosed in PCT Published PatentApplications PCT/US95/01570 and PCT/US97/19791, and U.S. Pat. No.6,194,388 B1 and U.S. Pat. No. 6,239,116 B1, issued Feb. 27, 2001 andMay 29, 2001 respectively. Exemplary sequences include but are notlimited to those disclosed in these latter applications and patents.

The C class immunostimulatory nucleic acids contain at least twodistinct motifs have unique and desirable stimulatory effects on cellsof the immune system. Some of these ODN have both a traditional“stimulatory” CpG sequence and a “GC-rich” or “B-cell neutralizing”motif. These combination motif nucleic acids have immune stimulatingeffects that fall somewhere between those effects associated withtraditional “class B” CpG ODN, which are strong inducers of B cellactivation and dendritic cell (DC) activation, and those effectsassociated with a more recently described class of immune stimulatorynucleic acids (“class A” CpG ODN) which are strong inducers of IFN-α andnatural killer (NK) cell activation but relatively poor inducers ofB-cell and DC activation. Krieg A M et al. (1995) Nature 374:546-9;Ballas Z K et al. (1996) J Immunol 157:1840-5; Yamamoto S et al. (1992)J Immunol 148:4072-6. While preferred class B CpG ODN often havephosphorothioate backbones and preferred class A CpG ODN have mixed orchimeric backbones, the C class of combination motif immune stimulatorynucleic acids may have either stabilized, e.g., phosphorothioate,chimeric, or phosphodiester backbones, and in some preferredembodiments, they have semi-soft backbones.

In one aspect the invention provides immune stimulatory nucleic acidsbelonging to this new class of combination motif immune-stimulatorynucleic acids. The B cell stimulatory domain is defined by a formula: 5′X₁DCGHX₂ 3′. D is a nucleotide other than C. C is cytosine. G isguanine. H is a nucleotide other than G.

X₁ and X₂ are any nucleic acid sequence 0 to 10 nucleotides long. X₁ mayinclude a CG, in which case there is preferably a T immediatelypreceding this CG. In some embodiments DCG is TCG. X₁ is preferably from0 to 6 nucleotides in length. In some embodiments X₂ does not containany poly G or poly A motifs. In other embodiments the immunostimulatorynucleic acid has a poly-T sequence at the 5′ end or at the 3′ end. Asused herein, “poly-A” or “poly-T” shall refer to a stretch of four ormore consecutive A's or T's respectively, e.g., 5′ AAAA 3′ or 5′ TTTT3′.

As used herein, “poly-G end” shall refer to a stretch of four or moreconsecutive G's, e.g., 5′ GGGG 3′, occurring at the 5′ end or the 3′ endof a nucleic acid. As used herein, “poly-G nucleic acid” shall refer toa nucleic acid having the formula 5′ X₁X₂GGGX₃X₄ 3′ wherein X₁, X₂, X₃,and X₄ are nucleotides preferably at least one of X₃ and X₄ is a G.

Some preferred designs for the B cell stimulatory domain under,thisformula comprise TTTTTCG, TCG, TTCG, TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT,TCGTCGT. The second motif of the nucleic acid is referred to as either Por N and is positioned immediately 5′ to X₁ or immediately 3′ to X₂.

N is a B-cell neutralizing sequence that begins with a CGG trinucleotideand is at least 10 nucleotides long. A B-cell neutralizing motifincludes at least one CpG sequence in which the CG is preceded by a C orfollowed by a G (Krieg A M et al. (1998) Proc Nad Acad Sci USA95:12631-12636) or is a CG containing DNA sequence in which the C of theCG is methylated. As used herein, “CpG” shall refer to a 5′ cytosine (C)followed by a 3′ guanine (G) and linked by a phosphate bond. At leastthe C of the 5′ CG 3′ must be unmethylated. Neutralizing motifs aremotifs which has some degree of immunostimulatory capability whenpresent in an otherwise non-stimulatory motif, but, which when presentin the context of other immunostimulatory motifs serve to reduce theimmunostimulatory potential of the other motifs.

P is a GC-rich palindrome containing sequence at least 10 nucleotideslong. As used herein, “palindrome” and, equivalently, “palindromicsequence” shall refer to an inverted repeat, i.e., a sequence such asABCDEE′D′C′B′A′ in which A and A′, B and B′, etc., are bases capable offorming the usual Watson-Crick base pairs.

As used herein, “GC-rich palindrome” shall refer to a palindrome havinga base composition of at least two-thirds G's and C's. In someembodiments the GC-rich domain is preferably 3′ to the “B cellstimulatory domain”. In the case of a 10-base long GC-rich palindrome,the palindrome thus contains at least 8 G's and C's. In the case of a12-base long GC-rich palindrome, the palindrome also contains at least 8G's and C's. In the case of a 14-mer GC-rich palindrome, at least tenbases of the palindrome are G's and C's. In some embodiments the GC-richpalindrome is made up exclusively of G's and C's.

In some embodiments the GC-rich palindrome has a base composition of atleast 81 percent G's and C's. In the case of such a 10-base long GC-richpalindrome, the palindrome thus is made exclusively of G's and C's. Inthe case of such a 12-base long GC-rich palindrome, it is preferred thatat least ten bases (83 percent) of the palindrome are G's and C's. Insome preferred embodiments, a 12-base long GC-rich palindrome is madeexclusively of G's and C's. In the case of a 14-mer GC-rich palindrome,at least twelve bases (86 percent) of the palindrome are G's and C's. Insome preferred embodiments, a 14-base long GC-rich palindrome is madeexclusively of G's and C's. The C's of a GC-rich palindrome can beunmethylated or they can be methylated.

In general this domain has at least 3 Cs and Gs, more preferably 4 ofeach, and most preferably 5 or more of each. The number of Cs and Gs inthis domain need not be identical. It is preferred that the Cs and Gsare arranged so that they are able to form a self-complementary duplex,or palindrome, such as CCGCGCGG. This may be interrupted by As or Ts,but it is preferred that the self-complementarity is at least partiallypreserved as for example in the motifs CGACGTTCGTCG (SEQ ID NO:) ______)or CGGCGCCGTGCCG (SEQ ID NO: ______). When complementarity is notpreserved, it is preferred that the non-complementary base pairs be TG.In a preferred embodiment there are no more than 3 consecutive basesthat are not part of the palindrome, preferably no more than 2, and mostpreferably only 1. In some embodiments the GC-rich palindrome includesat least one CGG trimer, at least one CCG trimer, or at least one CGCGtetramer. In other embodiments the GC-rich palindrome is notCCCCCCGGGGGG (SEQ ID NO: ______) or GGGGGGCCCCCC (SEQ ID NO: ______),CCCCCGGGGG (SEQ ID NO: ______) or GGGGGCCCCC (SEQ ID NO: ______).

At least one of the G's of the GC rich region may be substituted with aninosine (I). In some embodiments P includes more than one I.

In certain embodiments the immunostimulatory nucleic acid has one of thefollowing formulas 5′ NX₁DCGHX₂ 3′, 5′ X₁DCGHX₂N 3′, 5′ PX_(I)DCGHX₂ 3′,5′ X₁DCGHX₂P 3′, 5′ X₁DCGHX₂PX₃ 3′, 5′ X₁DCGHPX₃ 3′, 5′ DCGHX₂PX₃ 3′, 5′TCGHX₂PX₃ 3′, 5′ DCGHPX₃ 3′, or 5′ DCGHP 3′.

In other aspects the invention provides immune stimulatory nucleic acidswhich are defined by a formula: 5′ N₁PyGN₂P 3′. N₁ is any sequence 1 to6 nucleotides long. Py is a pyrimidine. G is guanine. N₂ is any sequence0 to 30 nucleotides long. P is a GC-rich palindrome containing sequenceat least 10 nucleotides long.

N₁ and N₂ may contain more than 50% pyrimidines, and more preferablymore than 50% T. N₁ may include a CG, in which case there is preferablya T immediately preceding this CG. In some embodiments N₁PyG is TCG(such as ODN 5376, which has a 5′ TCGG), and most preferably a TCGN₂,where N₂ is not G.

N₁PyGN₂P may include one or more inosine (I) nucleotides. Either the Cor the G in N1 may be replaced by inosine, but the CpI is preferred tothe IpG. For inosine substitutions such as IpG, the optimal activity maybe achieved with the use of a “semi-soft” or chimeric backbone, wherethe linkage between the IG or the CI is phosphodiester. N₁ may includeat least one CI, TCI, IG or TIG motif

In certain embodiments N₁PyGN₂ is a sequence selected from the groupconsisting of TTTTTCG, TCG, TTCG, TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT,and TCGTCGT.

Some non limiting examples of C-Class nucleic acids include:

SEQ ID NO Sequence 17 T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 18T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 19T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 20T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 21T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 22T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 23T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 24T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 25T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G

For facilitating uptake into cells, immunostimulatory nucleic acids,including CpG-containing oligonucleotides, are preferably in the rangeof 8 to 100 bases in length. However, nucleic acids of any size greaterthan 8 nucleotides (even many kb long) are capable of inducing an immuneresponse according to the invention if sufficient immunostimulatorymotifs are present, since larger nucleic acids are degraded intooligonucleotides inside of cells. Preferably the immunostimulatorynucleic acid is in the range of between 8 and 100 nucleotides in length.In some preferred embodiments the immunostimulatory nucleic acids isbetween 12 and 40 nucleotides in length. In more preferred embodimentsthe immunostimulatory nucleic acids is between 8 and 30 nucleotides inlength. In most preferred embodiments the immunostimulatory nucleicacids is between 8 and 24 nucleotides in length.

“Palindromic sequence” shall mean an inverted repeat, i.e., a sequencesuch as ABCDEE′D′C′B′A′ in which A and A′, B and B′, C and C′, D and D′,and E and E′ are bases capable of forming the usual Watson-Crick basepairs. In vivo, such palindromic sequences may form double-strandedstructures. In one embodiment the CpG oligonucleotide contains apalindromic sequence. A palindromic sequence used in this context refersto a palindrome in which the CpG is part of the palindrome, andpreferably is the center of the palindrome. In another embodiment theCpG oligonucleotide is free of a palindrome. A CpG oligonucleotide thatis free of a palindrome is one in which the CpG dinucleotide is not partof a palindrome. Such an oligonucleotide may include a palindrome inwhich the CpG is not the center of the palindrome.

In some embodiments of the invention the immunostimulatoryoligonucleotides include immunostimulatory motifs which are “CpGdinucleotides”. A CpG dinucleotide can be methylated or unmethylated. Animmunostimulatory nucleic acid containing at least one unmethylated CpGdinucleotide is a nucleic acid molecule which contains an unmethylatedcytosine-guanine dinucleotide sequence (i.e., an unmethylated 5′cytidine followed by 3′ guanosine and linked by a phosphate bond) andwhich activates the immune system; such an immunostimulatory nucleicacid is a CpG nucleic acid. CpG nucleic acids have been described in anumber of issued patents, published patent applications, and otherpublications, including U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806;6,218,371; 6,239,116; and 6,339,068. An immunostimulatory nucleic acidcontaining at least one methylated CpG dinucleotide is a nucleic acidwhich contains a methylated cytosine-guanine dinucleotide sequence(i.e., a methylated 5′ cytidine followed by a 3′ guanosine and linked bya phosphate bond) and which activates the immune system. In otherembodiments the immunostimulatory oligonucleotides are free of CpGdinucleotides. These oligonucleotides which are free of CpGdinucleotides are referred to as non-CpG oligonucleotides, and they havenon-CpG immunostimulatory motifs. The invention, therefore, alsoencompasses nucleic acids with other types of immunostimulatory motifs,which can be methylated or unmethylated. The immunostimulatoryoligonucleotides of the invention, further, can include any combinationof methylated and unmethylated CpG and non-CpG immunostimulatory motifs.

The immunostimulatory nucleic acid molecules may have a chimericbackbone. For purposes of the instant invention, a chimeric backbonerefers to a partially stabilized backbone, wherein at least oneinternucleotide linkage is phosphodiester or phosphodiester-like, andwherein at least one other internucleotide linkage is a stabilizedinternucleotide linkage, wherein the at least one phosphodiester orphosphodiester-like linkage and the at least one stabilized linkage aredifferent. Since boranophosphonate linkages have been reported to bestabilized relative to phosphodiester linkages, for purposes of thechimeric nature of the backbone, boranophosphonate linkages can beclassified either as phosphodiester-like or as stabilized, depending onthe context. For example, a chimeric backbone according to the instantinvention could in one embodiment include at least one phosphodiester(phosphodiester or phosphodiester-like) linkage and at least oneboranophosphonate (stabilized) linkage. In another embodiment a chimericbackbone according to the instant invention could includeboranophosphonate (phosphodiester or phosphodiester-like) andphosphorothioate (stabilized) linkages. A “stabilized internucleotidelinkage” shall mean an internucleotide linkage that is relativelyresistant to in vivo degradation (e.g., via an exo- or endo-nuclease),compared to a phosphodiester internucleotide linkage. Preferredstabilized internucleotide linkages include, without limitation,phosphorothioate, phosphorodithioate, methylphosphonate, andmethylphosphorothioate. Other stabilized internucleotide linkagesinclude, without limitation: peptide, alkyl, dephospho, and others asdescribed above.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g., asdescribed in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (inwhich the charged oxygen moiety is alkylated as described in U.S. Pat.No. 5,023,243 and European Patent No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described. Uhlmann E et al. (1990) Chem Rev 90:544; GoodchildJ (1990) Bioconjugate Chem 1:165. Methods for preparing chimericoligonucleotides are also known. For instance patents issued to Uhlmannet al have described such techniques.

Mixed backbone modified ODN may be synthesized using a commerciallyavailable DNA synthesizer and standard phosphoramidite chemistry. (F. E.Eckstein, “Oligonucleotides and Analogues—A Practical Approach” IRLPress, Oxford, UK, 1991, and M. D. Matteucci and M. H. Caruthers,Tetrahedron Lett. 21, 719 (1980)) After coupling, PS linkages areintroduced by sulfurization using the Beaucage reagent (R. P. Iyer, W.Egan, J. B. Regan and S. L. Beaucage, J. Am. Chem. Soc. 112, 1253(1990)) (0.075 M in acetonitrile) or phenyl acetyl disulfide (PADS)followed by capping with acetic anhydride, 2,6-lutidine intetrahydrofurane (1:1:8; v:v:v) and N-methylimidazole (16% intetrahydrofurane). This capping step is performed after thesulfurization reaction to minimize formation of undesired phosphodiester(PO) linkages at positions where a phosphorothioate linkage should belocated. In the case of the introduction of a phosphodiester linkage,e.g. at a CpG dinucleotide, the intermediate phosphorous-III is oxidizedby treatment with a solution of iodine in water/pyridine. After cleavagefrom the solid support and final deprotection by treatment withconcentrated ammonia (15 hrs at 50° C.), the ODN are analyzed by HPLC ona Gen-Pak Fax column (Millipore-Waters) using a NaCl-gradient (e.g.buffer A: 10 mM NaH₂PO₄ in acetonitrile/water=1:4/v:v pH 6.8; buffer B:10 mM NaH₂PO₄, 1.5 M NaCl in acetonitrile/water=1:4/v:v; 5 to 60% B in30 minutes at 1 ml/min) or by capillary gel electrophoresis. The ODN canbe purified by HPLC or by FPLC on a Source High Performance column(Amersham Pharmacia). HPLC-homogeneous fractions are combined anddesalted via a C18 column or by ultrafiltration. The ODN was analyzed byMALDI-TOF mass spectrometry to confirm the calculated mass.

The nucleic acids of the invention can also include other modifications.These include nonionic DNA analogs, such as alkyl- and aryl-phosphates(in which the charged phosphonate oxygen is replaced by an alkyl or arylgroup), phosphodiester and alkylphosphotriesters, in which the chargedoxygen moiety is alkylated. Nucleic acids which contain diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

In some embodiments the oligonucleotides may be soft or semi-softoligonucleotides. A soft oligonucleotide is an immunostimulatoryoligonucleotide having a partially stabilized backbone, in whichphosphodiester or phosphodiester-like internucleotide linkages occuronly within and immediately adjacent to at least one internalpyrimidine-purine dinucleotide (YZ). Preferably YZ is YG, apyrimidine-guanosine (YG) dinucleotide. The at least one internal YZdinucleotide itself has a phosphodiester or phosphodiester-likeinternucleotide linkage. A phosphodiester or phosphodiester-likeinternucleotide linkage occurring immediately adjacent to the at leastone internal YZ dinucleotide can be 5′, 3′, or both 5′ and 3′ to the atleast one internal YZ dinucleotide.

In particular, phosphodiester or phosphodiester-like internucleotidelinkages involve “internal dinucleotides”. An internal dinucleotide ingeneral shall mean any pair of adjacent nucleotides connected by aninternucleotide linkage, in which neither nucleotide in the pair ofnucleotides is a terminal nucleotide, i.e., neither nucleotide in thepair of nucleotides is a nucleotide defining the 5′ or 3′ end of theoligonucleotide. Thus a linear oligonucleotide that is n nucleotideslong has a total of n-1 dinucleotides and only n-3 internaldinucleotides. Each internucleotide linkage in an internal dinucleotideis an internal internucleotide linkage. Thus a linear oligonucleotidethat is n nucleotides long has a total of n-1 internucleotide linkagesand only n-3 internal internucleotide linkages. The strategically placedphosphodiester or phosphodiester-like internucleotide linkages,therefore, refer to phosphodiester or phosphodiester-likeinternucleotide linkages positioned between any pair of nucleotides inthe nucleic acid sequence. In some embodiments the phosphodiester orphosphodiester-like internucleotide linkages are not positioned betweeneither pair of nucleotides closest to the 5′ or 3′ end.

Preferably a phosphodiester or phosphodiester-like internucleotidelinkage occurring immediately adjacent to the at least one internal YZdinucleotide is itself an internal internucleotide linkage. Thus for asequence N₁ YZ N₂, wherein N₁ and N₂ are each, independent of the other,any single nucleotide, the YZ dinucleotide has a phosphodiester orphosphodiester-like internucleotide linkage, and in addition (a) N₁ andY are linked by a phosphodiester or phosphodiester-like internucleotidelinkage when N₁ is an internal nucleotide, (b) Z and N₂ are linked by aphosphodiester or phosphodiester-like internucleotide linkage when N₂ isan internal nucleotide, or (c) N₁ and Y are linked by a phosphodiesteror phosphodiester-like internucleotide linkage when N₁ is an internalnucleotide and Z and N₂ are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₂ is an internalnucleotide.

Soft oligonucleotides according to the instant invention are believed tobe relatively susceptible to nuclease cleavage compared to completelystabilized oligonucleotides. Without meaning to be bound to a particulartheory or mechanism, it is believed that soft oligonucleotides of theinvention are cleavable to fragments with reduced or noimmunostimulatory activity relative to full-length softoligonucleotides. Incorporation of at least one nuclease-sensitiveinternucleotide linkage, particularly near the middle of theoligonucleotide, is believed to provide an “off switch” which alters thepharmacokinetics of the oligonucleotide so as to reduce the duration ofmaximal immunostimulatory activity of the oligonucleotide. This can beof particular value in tissues and in clinical applications in which itis desirable to avoid injury related to chronic local inflammation orimmunostimulation, e.g., the kidney.

A semi-soft oligonucleotide is an immunostimulatory oligonucleotidehaving a partially stabilized backbone, in which phosphodiester orphosphodiester-like internucleotide linkages occur only within at leastone internal pyrimidine-purine (YZ) dinucleotide. Semi-softoligonucleotides generally possess increased immunostimulatory potencyrelative to corresponding fully stabilized immunostimulatoryoligonucleotides. Due to the greater potency of semi-softoligonucleotides, semi-soft oligonucleotides may be used, in someinstances, at lower effective concentations and have lower effectivedoses than conventional fully stabilized immunostimulatoryoligonucleotides in order to achieve a desired biological effect.

It is believed that the foregoing properties of semi-softoligonucleotides generally increase with increasing “dose” ofphosphodiester or phosphodiester-like internucleotide linkages involvinginternal YZ dinucleotides. Thus it is believed, for example, thatgenerally for a given oligonucleotide sequence with five internal YZdinucleotides, an oligonucleotide with five internal phosphodiester orphosphodiester-like YZ internucleotide linkages is moreimmunostimulatory than an oligonucleotide with four internalphosphodiester or phosphodiester-like YG internucleotide linkages, whichin turn is more immunostimulatory than an oligonucleotide with threeinternal phosphodiester or phosphodiester-like YZ internucleotidelinkages, which in turn is more immunostimulatory than anoligonucleotide with two internal phosphodiester or phosphodiester-likeYZ internucleotide linkages, which in turn is more immunostimulatorythan an oligonucleotide with one internal phosphodiester orphosphodiester-like YZ internucleotide linkage. Importantly, inclusionof even one internal phosphodiester or phosphodiester-like YZinternucleotide linkage is believed to be advantageous over no internalphosphodiester or phosphodiester-like YZ internucleotide linkage. Inaddition to the number of phosphodiester or phosphodiester-likeinternucleotide linkages, the position along the length of the nucleicacid can also affect potency.

The soft and semi-soft oligonucleotides will generally include, inaddition to the phosphodiester or phosphodiester-like internucleotidelinkages at preferred internal positions, 5′ and 3′ ends that areresistant to degradation. Such degradation-resistant ends can involveany suitable modification that results in an increased resistanceagainst exonuclease digestion over corresponding unmodified ends. Forinstance, the 5′ and 3′ ends can be stabilized by the inclusion there ofat least one phosphate modification of the backbone. In a preferredembodiment, the at least one phosphate modification of the backbone ateach end is independently a phosphorothioate, phosphorodithioate,methylphosphonate, or methylphosphorothioate internucleotide linkage. Inanother embodiment, the degradation-resistant end includes one or morenucleotide units connected by peptide or amide linkages at the 3′ end.

A phosphodiester internucleotide linkage is the type of linkagecharacteristic of nucleic acids found in nature. As shown in FIG. 20,the phosphodiester internucleotide linkage includes a phosphorus atomflanked by two bridging oxygen atoms and bound also by two additionaloxygen atoms, one charged and the other uncharged. Phosphodiesterinternucleotide linkage is particularly preferred when it is importantto reduce the tissue half-life of the oligonucleotide.

A phosphodiester-like internucleotide linkage is a phosphorus-containingbridging group that is chemically and/or diastereomerically similar tophosphodiester. Measures of similarity to phosphodiester includesusceptibility to nuclease digestion and ability to activate RNAse H.Thus for example phosphodiester, but not phosphorothioate,oligonucleotides are susceptible to nuclease digestion, while bothphosphodiester and phosphorothioate oligonucleotides activate RNAse H.In a preferred embodiment the phosphodiester-like internucleotidelinkage is boranophosphate (or equivalently, boranophosphonate) linkage.U.S. Pat. No. 5,177,198; U.S. Pat. No. 5,859,231; U.S. Pat. No.6,160,109; U.S. Pat. No. 6,207,819; Sergueev et al., (1998) J Am ChemSoc 120:9417-27. In another preferred embodiment the phosphodiester-likeinternucleotide linkage is diasteromerically pure Rp phosphorothioate.It is believed that diasteromerically pure Rp phosphorothioate is moresusceptible to nuclease digestion and is better at activating RNAse Hthan mixed or diastereomerically pure Sp phosphorothioate. Stereoisomersof CpG oligonucleotides are the subject of co-pending U.S. patentapplication Ser. No. 09/361,575 filed Jul. 27, 1999, and published PCTapplication PCT/US99/17100 (WO 00/06588). It is to be noted that forpurposes of the instant invention, the term “phosphodiester-likeinternucleotide linkage” specifically excludes phosphorodithioate andmethylphosphonate internucleotide linkages.

As described above the soft and semi-soft oligonucleotides of theinvention may have phosphodiester like linkages between C and G. Oneexample of a phosphodiester-like linkage is a phosphorothioate'linkagein an Rp conformation. Oligonucleotide p-chirality can have apparentlyopposite effects on the immune activity of a CpG oligonucleotide,depending upon the time point at which activity is measured. At an earlytime point of 40 minutes, the R_(p) but not the S_(P) stereoisomer ofphosphorothioate CpG oligonucleotide induces JNK phosphorylation inmouse spleen cells. In contrast, when assayed at a late time point of 44hr, the S_(P) but not the R_(p) stereoisomer is active in stimulatingspleen cell proliferation. This difference in the kinetics andbioactivity of the R_(p) and S_(P) stereoisomers does not result fromany difference in cell uptake, but rather most likely is due to twoopposing biologic roles of the p-chirality. First, the enhanced activityof the Rp stereoisomer compared to the Sp for stimulating immune cellsat early time points indicates that the Rp may be more effective atinteracting with the CpG receptor, TLR9, or inducing the downstreamsignalling pathways. On the other hand, the faster degradation of the RpPS-oligonucleotides compared to the Sp results in a much shorterduration of signalling, so that the Sp PS-oligonucleotides appear to bemore biologically active when tested at later time points.

A surprisingly strong effect is achieved by the p-chirality at the CpGdinucleotide itself. In comparison to a stereo-random CpGoligonucleotide the congener in which the single CpG dinucleotide waslinked in Rp was slightly more active, while the congener containing anSp linkage was nearly inactive for inducing spleen cell proliferation.

The size (i.e., the number of nucleotide residues along the length ofthe nucleic acid) of the immunostimulatory oligonucleotide may alsocontribute to the stimulatory activity of the oligonucleotide. Forfacilitating uptake into cells immunostimulatory oligonucleotidespreferably have a minimum length of 6 nucleotide residues. Nucleic acidsof any size greater than 6 nucleotides (even many kb long) are capableof inducing an immune response according to the invention if sufficientimmunostimulatory motifs are present, since larger nucleic acids aredegraded inside of cells. It is believed by the instant inventors thatsemi-soft oligonucleotides as short as 4 nucleotides can also beimmunostimulatory if they can be delivered to the interior of the cell.In certain preferred embodiments according to the instant invention, theimmunostimulatory oligonucleotides are between 4 and 100 nucleotideslong. In typical embodiments the immunostimulatory oligonucleotides arebetween 6 and 40 nucleotides long. In certain preferred embodimentsaccording to the instant invention, the immunostimulatoryoligonucleotides are between 6 and 19 nucleotides long.

The immunostimulatory oligonucleotides generally have a length in therange of between 4 and 100 and in some embodiments 10 and 40. The lengthmay be in the range of between 16 and 24 nucleotides.

The terms “nucleic acid” and “oligonucleotide” also encompass nucleicacids or oligonucleotides with substitutions or modifications, such asin the bases and/or sugars. For example, they include nucleic acidshaving backbone sugars that are covalently attached to low molecularweight organic groups other than a hydroxyl group at the 2′ position andother than a phosphate group or hydroxy group at the 5′ position. Thusmodified nucleic acids may include a 2′-O-alkylated ribose group. Inaddition, modified nucleic acids may include sugars such as arabinose or2′-fluoroarabinose instead of ribose. Thus the nucleic acids may beheterogeneous in backbone composition thereby containing any possiblecombination of polymer units linked together such as peptide-nucleicacids (which have an amino acid backbone with nucleic acid bases).

Nucleic acids also include substituted purines and pyrimidines such asC-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases.Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,thymine, 5-methylcytosine, 5-hydroxycytosine, 5-fluorocytosine,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,and other naturally and non-naturally occurring nucleobases, substitutedand unsubstituted aromatic moieties. Other such modifications are wellknown to those of skill in the art.

The immunostimulatory oligonucleotides of the instant invention canencompass various chemical modifications and substitutions, incomparison to natural RNA and DNA, involving a phosphodiesterinternucleotide bridge, a β-D-ribose unit and/or a natural nucleotidebase (adenine, guanine, cytosine, thymine, uracil). Examples of chemicalmodifications are known to the skilled person and are described, forexample, in Uhlmann E et al. (1990) Chem Rev 90:543; “Protocols forOligonucleotides and Analogs” Synthesis and Properties & Synthesis andAnalytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993;Crooke S T et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; andHunziker J et al. (1995) Mod Synth Methods 7:331-417. An oligonucleotideaccording to the invention may have one or more modifications, whereineach modification is located at a particular phosphodiesterinternucleotide bridge and/or at a particular β-D-ribose unit and/or ata particular natural nucleotide base position in comparison to anoligonucleotide of the same sequence which is composed of natural DNA orRNA.

For example, the invention relates to an oligonucleotide which maycomprise one or more modifications and wherein each modification isindependently selected from:

-   a) the replacement of a phosphodiester internucleotide bridge    located at the 3′ and/or the 5′ end of a nucleotide by a modified    internucleotide bridge,-   b) the replacement of phosphodiester bridge located at the 3′ and/or    the 5′ end of a nucleotide by a dephospho bridge,-   c) the replacement of a sugar phosphate unit from the sugar    phosphate backbone by another unit,-   d) the replacement of a β-D-ribose unit by a modified sugar unit,    and-   e) the replacement of a natural nucleotide base by a modified    nucleotide base.

More detailed examples for the chemical modification of anoligonucleotide are as follows.

A phosphodiester internucleotide bridge located at the 3′ and/or the 5′end of a nucleotide can be replaced by a modified internucleotidebridge, wherein the modified internucleotide bridge is for exampleselected from phosphorothioate, phosphorodithioate,NR¹R²-phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate,phosphate-(C₁-C₂₁)-O-alkyl ester,phosphate-[(C₆-C₁₂)aryl-(C₁-C₂₁)-O-alkyl]ester, (C₁-C₈)alkylphosphonateand/or (C₆-C₁₂)arylphosphonate bridges, (C₇-C₁₂)-α-hydroxymethyl-aryl(e.g., disclosed in WO 95/01363), wherein (C₆-C₁₂)aryl, (C₆-C₂₀)aryl and(C₆-C₁₄)aryl are optionally substituted by halogen, alkyl, alkoxy,nitro, cyano, and where R¹ and R² are, independently of each other,hydrogen, (C₁-C₁₈)-alkyl, (C₆-C₂₀)-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl,preferably hydrogen, (C₁-C₈)-alkyl, preferably (C₁-C₄)-alkyl and/ormethoxyethyl, or R¹ and R² form, together with the nitrogen atomcarrying them, a 5-6-membered heterocyclic ring which can additionallycontain a further heteroatom from the group O, S and N.

The replacement of a phosphodiester bridge located at the 3′ and/or the5′ end of a nucleotide by a dephospho bridge (dephospho bridges aredescribed, for example, in Uhlmann E and Peyman A in “Methods inMolecular Biology”, Vol. 20, “Protocols for Oligonucleotides andAnalogs”, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp.355 ff), wherein a dephospho bridge is for example selected from thedephospho bridges formacetal, 3′-thioformacetal, methylhydroxylamine,oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silylgroups.

A sugar phosphate unit (i.e., a β-D-ribose and phosphodiesterinternucleotide bridge together forming a sugar phosphate unit) from thesugar phosphate backbone (i.e., a sugar phosphate backbone is composedof sugar phosphate units) can be replaced by another unit, wherein theother unit is for example suitable to build up a “morpholino-derivative”oligomer (as described, for example, in Stirchak E P et al. (1989)Nucleic Acids Res 17:6129-41), that is, e.g., the replacement by amorpholino-derivative unit; or to build up a polyamide nucleic acid(“PNA”; as described for example, in Nielsen P E et al. (1994) BioconjugChem 5:3-7), that is, e.g., the replacement by a PNA backbone unit,e.g., by 2-aminoethylglycine.

A β-ribose unit or a β-D-2′-deoxyribose unit can be replaced by amodified sugar unit, wherein the modified sugar unit is for exampleselected from β-D-ribose, α-D-2′-deoxyribose, L-2′-deoxyribose,2′-F-2′-deoxyribose, 2′-F-arabinose, 2-O—(C₁-C₆)alkyl-ribose, preferably2′-O—(C₁-C₆)alkyl-ribose is 2′-O-methylribose,2′-O—(C₂-C₆)alkenyl-ribose, 2′-[O—(C₁-C₆)alkyl-O—(C₁-C₆)alkyl]-ribose,2′-NH₂-2′-deoxyribose, β-D-xylo-furanose, α-arabinofuranose,2,4-dideoxy-β-D-erythro-hexo-pyranose, and carbocyclic (described, forexample, in Froehler J (1992) Am Chem Soc 114:8320) and/or open-chainsugar analogs (described, for example, in Vandendriessche et al. (1993)Tetrahedron 49:7223) and/or bicyclosugar analogs (described, forexample, in Tarkov M et al. (1993) Helv Chim Acta 76:481).

In some preferred embodiments the sugar is 2′-O-methylribose,particularly for one or both nucleotides linked by a phosphodiester orphosphodiester-like internucleotide linkage.

Nucleic acids also include substituted purines and pyrimidines such asC-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases.Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,and thymine, and other naturally and non-naturally occurringnucleobases, substituted and unsubstituted aromatic moieties.

A modified base is any base which is chemically distinct from thenaturally occurring bases typically found in DNA and RNA such as T, C,G, A, and U, but which share basic chemical structures with thesenaturally occurring bases. The modified nucleotide base may be, forexample, selected from hypoxanthine, uracil, dihydrouracil,pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-(C₁-C₆)-alkyuracil, 5-(C₂-C₆)-alkenyluracil, 5-(C₂-C₆)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C₁-C₆)-alkylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N²-dimethylguanine, 2,4-diamino-purine, 8-azapurine, asubstituted 7-deazapurine, preferably 7-deaza-7-substituted and/or7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine,e.g., N4-ethylcytosine, 5-hydroxydeoxycytidine,5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g.,N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and deoxyribonucleotides ofnitropyrrole, C5-propynylpyrimidine, and diaminopurine e.g.,2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, hypoxanthine or other modifications of a naturalnucleotide bases. This list is meant to be exemplary and is not to beinterpreted to be limiting.

In particular formulas described herein a set of modified bases isdefined. For instance the letter Y is used to refer to a nucleotidecontaining a cytosine or a modified cytosine. A modified cytosine asused herein is a naturally occurring or non-naturally occurringpyrimidine base analog of cytosine which can replace this base withoutimpairing the immunostimulatory activity of the oligonucleotide.Modified cytosines include but are not limited to 5-substitutedcytosines (e.g. 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine,5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstitutedor substituted 5-alkynyl-cytosine), 6-substituted cytosines,N4-substituted cytosines (e.g. N4-ethyl-cytosine), 5-aza-cytosine,2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogswith condensed ring systems (e.g. N,N′-propylene cytosine orphenoxazine), and uracil and its derivatives (e.g. 5-fluoro-uracil,5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil,5-propynyl-uracil). Some of the preferred cytosines include5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodimentof the invention, the cytosine base is substituted by a universal base(e.g. 3-nitropyrrole, P-base), an aromatic ring system (e.g.fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer).

The letter Z is used to refer to guanine or a modified guanine base. Amodified guanine as used herein is a naturally occurring ornon-naturally occurring purine base analog of guanine which can replacethis base without impairing the immunostimulatory activity of theoligonucleotide. Modified guanines include but are not limited to7-deazaguanine, 7-deaza-7-substituted guanine (such as7-deaza-7-(C2-C6)alkynylguanine), 7-deaza-8-substituted guanine,hypoxanthine, N2-substituted guanines (e.g. N2-methyl-guanine),5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione,2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substitutedadenines (e.g. N6-methyl-adenine, 8-oxo-adenine) 8-substituted guanine(e.g. 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. Inanother embodiment of the invention, the guanine base is substituted bya universal base (e.g. 4-methyl-indole, 5-nitro-indole, and K-base), anaromatic ring system (e.g. benzimidazole or dichloro-benzimidazole,1-methyl-1H-[1,2,4]triazole-3-carboxylic acid amide) or a hydrogen atom(dSpacer).

The oligonucleotides may have one or more accessible 5′ ends. It ispossible to create modified oligonucleotides having two such 5′ ends.This may be achieved, for instance by attaching two oligonucleotidesthrough a 3′-3′ linkage to generate an oligonucleotide having one or twoaccessible 5′ ends. The 3′3′-linkage may be a phosphodiester,phosphorothioate or any other modified internucleotide bridge. Methodsfor accomplishing such linkages are known in the art. For instance, suchlinkages have been described in Seliger, H.; et al., Oligonucleotideanalogs with terminal 3′-3′- and 5′-5′-internucleotidic linkages asantisense inhibitors of viral gene expression, Nucleotides & Nucleotides(1991), 10(1-3), 469-77 and Jiang, et al., Pseudo-cyclicoligonucleotides: in vitro and in vivo properties, Bioorganic &Medicinal Chemistry (1999), 7(12), 2727-2735.

Additionally, 3′3′-linked nucleic acids where the linkage between the3′-terminal nucleotides is not a phosphodiester, phosphorothioate orother modified bridge, can be prepared using an additional spacer, suchas tri- or tetra-ethylenglycol phosphate moiety (Durand, M. et al,Triple-helix formation by an oligonucleotide containing one (dA)12 andtwo (dT)12 sequences bridged by two hexaethylene glycol chains,Biochemistry (1992), 31(38), 9197-204, U.S. Pat. No. 5,658,738, and U.S.Pat. No. 5,668,265). Alternatively, the non-nucleotidic linker may bederived from ethanediol, propanediol, or from an abasic deoxyribose(dSpacer) unit (Fontanel, Marie Laurence et al., Sterical recognition byT4 polynucleotide kinase of non-nucleosidic moieties 5′-attached tooligonucleotides; Nucleic Acids Research (1994), 22(11), 2022-7) usingstandard phosphoramidite chemistry. The non-nucleotidic linkers can beincorporated once or multiple times, or combined with each otherallowing for any desirable distance between the 3′-ends of the two ODNsto be linked.

For use in the instant invention, the oligonucleotides of the inventioncan be synthesized de novo using any of a number of procedures wellknown in the art. For example, the b-cyanoethyl phosphoramidite method(Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981);nucleotide H-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054,1986; Froehler et al., Nucl. Acid. Res. 14:5399-5407, 1986,; Garegg etal., Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let.29:2619-2622, 1988). These chemistries can be performed by a variety ofautomated nucleic acid synthesizers available in the market. Theseoligonucleotides are referred to as synthetic oligonucleotides. Anisolated oligonucleotide generally refers to an oligonucleotide which isseparated from components which it is normally associated with innature. As an example, an isolated oligonucleotide may be one which isseparated from a cell, from a nucleus, from mitochondria or fromchromatin.

The oligonucleotides are partially resistant to degradation (e.g., arestabilized).

A “stabilized oligonucleotide molecule” shall mean an oligonucleotidethat is relatively resistant to in vivo degradation (e.g. via an exo- orendo-nuclease). Nucleic acid stabilization can be accomplished viabackbone modifications. Oligonucleotides having phosphorothioatelinkages provide maximal activity and protect the oligonucleotide fromdegradation by intracellular exo- and endo-nucleases. Other modifiedoligonucleotides include phosphodiester modified nucleic acids,combinations of phosphodiester and phosphorothioate nucleic acid,methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy,and combinations thereof.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl-and alkyl-phosphonates can be made, e.g., as describedin U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which thecharged oxygen moiety is alkylated as described in U.S. Pat. No.5,023,243 and European Patent No. 092,574) can be prepared by automatedsolid phase synthesis using commercially available reagents. Methods formaking other DNA backbone modifications and substitutions have beendescribed (e.g., Uhlmann, E. and Peyman, A., Chem. Rev. 90:544, 1990;Goodchild, J., Bioconjugate Chem. 1:165, 1990).

Other stabilized oligonucleotides include: nonionic DNA analogs, such asalkyl- and aryl-phosphates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Nucleic acids which contain diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

The immunostimulatory oligonucleotides may also contain one or moreunusual linkages between the nucleotide or nucleotide-analogousmoieties. The usual internucleoside linkage is the 3′5′-linkage. Allother linkages are considered as unusual internucleoside linkages, suchas 2′5′-, 5′5′-, 3′3′-, 2′2′-, 2′3′-linkages. Thereby, the nomenclature2′ to 5′ is chosen according to the carbon atom of ribose. However, ifunnatural sugar moieties are employed, such as ring-expanded sugaranalogs (e.g. hexanose, cylohexene or pyranose) or bi- or tricyclicsugar analogs, then this nomenclature changes according to thenomenclature of the monomer. In 3′-deoxy-β-D-ribopyranose analogs (alsocalled p-DNA), the mononucleotides are e.g. connected via a4′2′-linkage.

If the nucleotide contains one 3′3′-linkage, then this oligonucleotideanalog will have two unlinked 5′-ends. Similarly, if the nucleotidecontains one 5′5′-linkage, then this oligonucleotide analog will havetwo unlinked 3′-ends. The accessibility of unlinked ends of nucleotidesmay be better accessible by their receptors. Both types of unusuallinkages (3′3′- and 5′5′) were described by Ramalho Ortigao et al.(Antisense Research and Development (1992) 2, 129-46), wherebyoligonucleotides having a 3′3′-linkage were reported to show enhancedstability towards cleavage by nucleases.

Different types of linkages can also be combined in one molecule whichmay lead to branching of the oligomer. If one part of theoligonucleotide is connected at the 3′-end via a 3′3′-linkage to asecond oligonucleotide part and at the 2′-end via a 2′3′-linkage to athird part of the molecule, this results e.g. in a branchedoligonucleotide with three 5′-ends (3′3′-, 2′3′-branched).

In principle, linkages between different parts of an oligonucleotide orbetween different oligonucleotides, respectively, can occur via allparts of the molecule, as long as this does not negatively interferewith the recognition by its receptor. According to the nature of thenucleic acid, the linkage can involve the sugar moiety (Su), theheterocyclic nucleobase (Ba) or the phosphate backbone (Ph). Thus,linkages of the type Su-Su, Su-Ph, Su-Ba, Ba—Ba, Ba-Su, Ba-Ph, Ph-Ph,Ph-Su, and Ph-Ba are possible. If the oligonucleotides are furthermodified by certain non-nucleotidic substituents, the linkage can alsooccur via the modified parts of the oligonucleotides. Thesemodifications include also modified nucleic acids, e.g. PNA, LNA, orMorpholino Oligonucleotide analogs.

The linkages are preferably composed of C, H, N, O, S, B, P, andHalogen, containing 3 to 300 atoms. An example with 3 atoms is an acetallinkage (ODN1-3′-O—CH₂—O-3′-ODN2; Froehler and Matteucci) connectinge.g. the 3′-hydroxy group of one nucleotide to the 3′-hydroxy group of asecond oligonucleotide. An example with about 300 atoms is PEG-40(tetraconta polyethyleneglycol). Preferred linkages are phosphodiester,phosphorothioate, methylphosphonate, phosphoramidate, boranophosphonate,amide; ether, thioether, acetal, thioacetal, urea, thiourea,sulfonamide, Schiff' Base and disulfide linkages. Another possibility isthe use of the Solulink BioConjugation System (www.trilinkbiotech.com).

If the oligonucleotide is composed of two or more sequence parts, theseparts can be identical or different. Thus, in an oligonucleotide with a3′3′-linkage, the sequences can be identical 5′-ODN1-3′3′-ODN1-5′ ordifferent 5′-ODN1-3′3′-ODN2-5′. Furthermore, the chemical modificationof the various oligonucleotide parts as well as the linker connectingthem may be different. Since the uptake of short oligonucleotidesappears to be less efficient than that of long oligonucleotides, linkingof two or more short sequences results in improved immune stimulation.The length of the short oligonucleotides is preferably 2-20 nucleotides,more preferably 3-16 nucleotides, but most preferably 5-10 nucleotides.Preferred are linked oligonucleotides which have two or more unlinked5′-ends.

The oligonucleotide partial sequences may also be linked bynon-nucleotidic linkers, in particular abasic linkers (dSpacers),trietyhlene glycol units or hexaethylene glycol units. Further preferredlinkers are alkylamino linkers, such as C3, C6, C12 aminolinkers, andalso alkylthiol linkers, such as C3 or C6 thiol linkers. Theoligonucleotides can also be linked by aromatic residues which may befurther substituted by alkyl or substituted alkyl groups. Theoligonucleotides may also contain a Doubler or Trebler unit(www.glenres.com), in particular those oligonucleotides with a3′3′-linkage. Branching of the oligonucleotides by multiple doubler,trebler, or other multiplier units leads to dendrimers which are afurther embodiment of this invention. The oligonucleotides may alsocontain linker units resulting from peptide modifying reagents oroligonucleotide modifying reagents (www.glenres.com). Furthermore, itmay contain one or more natural or unnatural amino acid residues whichare connected by peptide (amide) linkages.

Another possibility for linking oligonucleotides is via crosslinking ofthe heterocyclic bases (Verma and Eckstein; Annu. Rev. Biochem. (1998)67: 99-134; page 124). Yet another possibility is a linkage between thesugar moiety of one sequence part with the heterocyclic base of anothersequence part (Iyer et al. Curr. Opin. Mol. Therapeutics (1999) 1:344-358; page 352).

The different oligonucleotides are synthesized by established methodsand can be linked together on-line during solid-phase synthesis.Alternatively, they may be linked together post-synthesis of theindividual partial sequences.

A “subject” shall mean a human or vertebrate animal including but notlimited to a dog, cat, horse, cow, pig, sheep, goat, chicken, non-humanprimate (e.g., monkey), fish (aquaculture species, e.g., salmon),rabbit, rat, and mouse.

A “subject having a viral infection” is a subject that has been exposedto a virus and has acute or chronic manifestations or detectable levelsof the virus in the body. In preferred embodiments of the invention, thesubject is one having a chronic viral infection, more preferably achronic hepatitis C infection. In important aspects of the invention,the subject is one that is non-responsive to prior therapy for hepatitisC infection. For example, a non-responsive subject includes one that waspreviously treated for hepatitis C infection with, for example, IFN-α(e.g., Intron A), and but such treatment was not successful, asdescribed herein. The invention intends to treat subjects that arenon-responsive, and in some instances to identify subjects that would benon-responsive in order to triage effective treatment.

Immunostimulatory nucleic acids can be effective in any vertebrate.Different immunostimulatory nucleic acids can cause optimal immunestimulation depending on the mammalian species. Thus animmunostimulatory nucleic acid causing optimal stimulation or inhibitionin humans may not cause optimal stimulation or inhibition in a mouse,and vice versa. One of skill in the art can identify the mostappropriate immunostimulatory nucleic acids useful for a particularmammalian species of interest using routine assays described hereinand/or known in the art, using the guidance supplied herein.

The immunostimulatory nucleic acid may be directly administered to thesubject or may be administered in-conjunction with a nucleic aciddelivery complex. A “nucleic acid delivery complex” shall mean a nucleicacid molecule associated with (e.g., ionically or covalently bound to,or encapsulated within) a targeting means (e.g., a molecule that resultsin higher affinity binding to target cell (e.g., pDCs or B cells) and/orincreased cellular uptake by target cells. Examples of nucleic aciddelivery complexes include nucleic acids associated with: a sterol(e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome orliposome), or a target cell specific binding agent (e.g., a ligandrecognized by target cell specific receptor). Preferred complexes may besufficiently stable in vivo to prevent significant uncoupling prior tointernalization by the target cell. However, the complex can becleavable under appropriate conditions within the cell so that thenucleic acid is released in a functional form.

The immunostimulatory nucleic acid or other therapeutics may beadministered alone (e.g., in saline or buffer) or using any deliveryvehicles known in the art. For instance the following delivery vehicleshave been described: cochleates; emulsomes; ISCOMs; liposomes; livebacterial vectors (e.g., Salmonella, Escherichia coli, BacillusCalmette-Guerin, Shigella, Lactobacillus); live viral vectors (e.g.,Vaccinia, adenovirus, Herpes Simplex); microspheres; nucleic acidvaccines; polymers (e.g., carboxymethylcellulose, chitosan); polymerrings; Proteosomes; sodium fluoride; transgenic plants; virosomes;virus-like particles. Those skilled in the art will recognize that otherdelivery vehicles that are known in the art may also be used.

Combined with the teachings provided herein, by choosing among thevarious active compounds and weighing factors such as potency, relativebioavailability, patient body weight, severity of adverse side-effectsand preferred mode of administration, an effective therapeutic treatmentregimen can be planned which does not cause substantial toxicity and yetis entirely effective to treat the particular subject as describedabove. The effective amount for any particular application can varydepending on such factors as the disease or condition being treated, theparticular immunostimulatory nucleic acid being administered (e.g., theclass of CpG immunostimulatory nucleic acid, the number of unmethylatedCpG motifs or their location in the nucleic acid, the degree ofchirality to the oligonucleotide, etc.), whether an antigen is alsoadministered and the nature of such antigen, the size of the subject, orthe severity of the disease or condition. One of ordinary skill in theart can empirically determine the effective amount of a particularimmunostimulatory nucleic acids and/or other therapeutic agent withoutnecessitating undue experimentation.

For adult human subjects, doses of the immunostimulatory nucleic acidscompounds described herein typically range from about 50 μg/dose to 20mg/dose, more typically from about 80 μg/dose to 8 mg/dose, and mosttypically from about 800 μg/dose to 4 mg/dose. Stated in terms ofsubject body weight, typical dosages range from about 0.5 to 500μg/kg/dose, more typically from about 1 to 100 μg/kg/dose, and mosttypically from about 10 to 50 μg/kg/dose. Doses will depend on factorsincluding the route of administration, e.g., oral administration mayrequire a substantially larger dose than subcutaneous administration.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

The immunostimulatory nucleic acids can be given in conjunction withother agents known in the art to be useful in treating viral infections.Of particular importance is the combination of immunostimulatory nucleicacids with anti-viral agents such as IFN-α, as demonstrated in theExamples section, to provide a synergistic response. Immunostimulatorynucleic acids can be used as a substitute for Ribavirin, which currentlyis administered together with IFN-α. Examples of such other agentscurrently used or under investigation for use in combination with IFN-αinclude amantadine, and cytokines, including IL-2, IL-10, IL-12, andIFN-γ.

Antiviral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g., amantadine), synthesisor translation of viral mRNA, including translation initiation (e.g.,interferon, antisense, and ribozymes), virus enzymes (e.g.,nonstructural serine proteases, RNA polymerases, reverse transcriptasesand helicases), replication of viral RNA or DNA (e.g., nucleosideanalogues), maturation of new virus proteins (e.g., protease inhibitorssuch as serine protease inhibitor BILN2061ZW from Boehringer Ingelheim),anti-oxidants such as Livfit (U.S. Pat. No. 6,136,316), and budding andrelease of the virus. Other anti-viral agents are described in U.S. Pat.Nos. 6,130,326, and 6,440,985, and published US patent application20020095033. Ribavirin analogues are also anti-viral agents embraced bythe invention.

Nucleotide analogues are synthetic compounds which are similar tonucleotides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleotide analogues are in the cell, they arephosphorylated, producing the triphosphate formed which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleotide analogue is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination.

Immunoglobulin therapy is typically used for the prevention of viralinfection, but can also be used to reduce levels of circulating virusand preventing newly formed cells from becoming infected. Immunoglobulintherapy for viral infections is different than bacterial infections,because rather than being antigen-specific, the immunoglobulin therapyfunctions by binding to extracellular virions and preventing them fromattaching to and entering cells which are susceptible to the viralinfection. The therapy is useful for the reduction of viremia for theperiod of time that the antibodies are present in the host. In generalthere are two types of immunoglobulin therapies, normal immunoglobulintherapy and hyper-immunoglobulin therapy. Normal immune globulin therapyutilizes an antibody product which is prepared from the serum of normalblood donors and pooled. This pooled product contains low titers ofantibody to a wide range of human viruses, such as hepatitis A,parvovirus, enterovirus (especially in neonates). To use normal immuneglobulin therapy for HCV, the serum would have to be obtained frompeople who were previously infected with HCV and who have successfullycleared the infection, either spontaneously or with some form oftherapy. Hyper-immune globulin therapy utilizes antibodies which areprepared from the serum of individuals who have high titers of anantibody to a particular virus. Those antibodies are then used against aspecific virus. For HCV, hyper-immune globulins could be produced byvaccinating volunteers with recombinant HCV proteins to producehepatitis C immune globulin.

Other anti-virals suitable in the methods of the invention aremanufactured by Triangle Pharmaceuticals, Inc., Gilead, ICN, Procter andGamble and ViroPharma Incorporated.

For use in therapy, an effective amount of the immunostimulatory nucleicacid can be administered to a subject by any mode that delivers theimmunostimulatory nucleic acids to the desired site, e.g., mucosal,systemic. “Administering” the pharmaceutical composition of the presentinvention may be accomplished by any means known to the skilled artisan.Preferred routes of administration include but are not limited to oral,parenteral, intralesional, topical, transdermal, intramuscular,intranasal, intratracheal, inhalational, ocular, vaginal, and rectal.

For oral administration, the compounds (i.e., immunostimulatory nucleicacids, or other therapeutic agents) can be formulated readily bycombining with pharmaceutically acceptable carriers well known in theart. Such carriers enable the compounds of the invention to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject tobe treated. Pharmaceutical preparations for oral use can be obtained assolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients are, inparticular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Optionally the oral formulations may also be formulated insaline or buffers for neutralizing internal acid conditions or may beadministered without any carriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer Science 249:1527 (1990),which is incorporated herein by reference.

The immunostimulatory nucleic acids may be administered per se (neat) orin the form of a pharmaceutically acceptable salt. When used in medicinethe salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic,formic, malonic, succinic, naphthalene-2-sulphonic, and benzenesulphonic. Also, such salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2 percentw/v); citric acid and a salt (1-3 percent w/v); boric acid and a salt(0.5-2.5 percent w/v); and phosphoric acid and a salt (0.8-2 percentw/v). Suitable preservatives include benzalkonium chloride (0.003-0.03percent w/v); chlorobutanol (0.3-0.9 percent w/v); parabens (0.01-0.25percent w/v) and thimerosal (0.004-0.02 percent w/v).

The pharmaceutical compositions of the invention contain apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the compounds of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular adjuvants orantigen selected, the particular condition being treated and the dosagerequired for therapeutic efficacy. The methods of this invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of an immune response without causing clinically unacceptableadverse effects. Preferred modes of administration are discussed above.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the compounds into associationwith a carrier which constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the compounds into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct. Liquid dose units are vials or ampoules. Solid dose units aretablets, capsules and suppositories. For treatment of a patient,depending on activity of the compound, manner of administration, purposeof the immunization (i.e., prophylactic or therapeutic), nature andseverity of the disorder, age and body weight of the patient, differentdoses may be necessary. The administration of a given dose can becarried out both by single administration in the form of an individualdose unit or else several smaller dose units.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They includepolymer-based systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-, di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which an agent of the invention iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775, 4,675,189; and 5,736,152, and (b) diffusional systems inwhich an active component permeates at a controlled rate from a polymersuch as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

In some embodiments, the immunostimulatory nucleic acid is modified. Incertain embodiments, the immunostimulatory nucleic acid has a modifiedbackbone with at least one nuclease-resistant internucleotide linkage. Anuclease-resistant internucleotide linkage can be selected from thegroup which includes a phosphorothioate linkage, a phosphorodithioatelinkage, a methylphosphonate linkage, and a peptide linkage. In certainembodiments a modified immunostimulatory nucleic acid includes at leastone nucleotide analog or at least one nucleotide analog. Theimmunostimulatory nucleic acid is a palindrome in certain embodiments,while in other embodiments, the immunostimulatory nucleic acid is not apalindrome. In some preferred embodiments the immunostimulatory nucleicacid is between 8 and 100 nucleotides in length, while in otherpreferred embodiments the immunostimulatory nucleic acid is between 12and 40 nucleotides in length. Preferred sizes, sequences andmodifications are described in greater detail below.

The following examples are included for purposes of illustration and arenot intended to limit the scope of the invention.

EXAMPLES

The purpose of this study was to evaluate the ability of differentclasses of CpG ODN to stimulate PBMC from HCV chronic carriers. PBMCwere isolated from whole blood collected from normal, healthy volunteersand chronic carriers of HCV and the ability of the different classes CpGODNs as well as soft and semi-soft molecules to stimulate B cellproliferation, cytokine secretion (IFN-g, TNF-α, IL-10 and IFN-α) andchemokine secretion (IP-10) in vitro was evaluated.

Also evaluated were the immune stimulatory effects of exogenous IFN-α-2b(Intron A) and Ribavirin, either alone, in combination with each other,and in combination with CpG ODN (B and C classes).

Materials and Methods Oligonucleotides

All oligonucleotide stocks were resuspended in TE buffer at pH 8.0(OmniPer®; EM Science, Gibbstown, N.J.). Dilutions of various ODNs weremade in RPMI 1640 complete media (Gibco BRL, Grand Island, N.Y.)containing 10% heat inactivated, normal human AB serum (Wisent Inc, St.Bruno, QC) and 1% penicillin/streptomycin (Gibco BRL, Grand Island,N.Y.) just prior to their use in cell assays. For the exogenous IFN-αsynergy experiments, Intron A (Interferon Alfa-2b, DIN 02223406,Schering Canada Inc., Pointe-Claire, Quebec, Canada) was added to theODN solutions to give final concentrations of 125 or 1000 IU/ml.Ribavirin (CAS 36791-04-5, Calbiochem, CN Biosciences Inc., La Jolla,Calif., USA) was reconstituted with sterile distilled water to produce a500 μm stock and diluted in media as described above, to give a finalconcentration of 5 μm in wells. Cells were incubated at 37° C. with 5%CO₂. After 48 h, cell supernatants were collected from each well andfrozen at −80° C.

ODNs used in experiments are shown in the following table:

TABLE 2 Sequences of oligos used in experiments SEQ ID NO: CLASSSEQUENCE 1 A G-G-GGACGACGTCGTGG-G-G-G-G-G 2 BTCG TCG TTT TGT CGT TTT GTC GTT 3 Control forTGC TGC TTT TTG CTG GCT TTT T B Class 4 C TCGTCGTTTTCGGCGGCCGCCG 5 BTCGTCGTTTCGTCGTTTTGTCGTT 6 B TCG TCG TTT TTC GTG CGT TTT T 7 Soft CTCGTCGTTT-T-C-G-G-CGGCCGCCG 8 semi-soft B TC-GTC-GTTTT-GTC-GTTTTGTC-GTT9 Semi-soft C TCGTC-GTTTTCGGC-GGCCGCCG 10 Semi-soft CTCGTCGTTTTC-GGCGGCC-GCCG 11 Semi-soft C TCGTCG-TTTTC-GGCGCGC-GCCG 12Semi-soft C TCGTC-GTTTTC-GGC-GCGC-GCCG 13 Semi-soft CTCGTCGTTTTAC-GGC-GCC-GTGCCG 14 Semi-soft C TCGTCG-TTTTAC-GGCGCC-GTGCCG15 Semi-soft C TCGTC-GTTTTAC-GGCGCC-GTGCCG 16 Semi-soft CTCGTC-GTTTTC-GGCGGCC-GCCG * A phosphodiester bond replacing aphosphorothioate bond within the oligonucleotide backbone is indicatedby (-)

Isolation of PBMCs

Whole blood (200 ml) was collected by venous puncture into heparinizedgreen top vacutainers from ten (10) normal, healthy, adult subjects andfifteen (15) adult subjects chronically infected with HCV who had aprevious 6 month course of an IFN-α-based therapy and were either atreatment failure or a relapsed responder. Peripheral blood mononuclearcells (PBMCs) were purified by centrifugation over Ficoll-Hypaque(Amersham Pharmacia Biotech, Uppsala, Sweden) at 400×g for 35 min. Cellswere resuspended at a concentration of 10×10⁶/ml in RPMI complete mediacontaining 10% normal human AB serum (heat inactivated) and 1%penicillin/streptomycin.

B-cell Proliferation

Cells were isolated as described above and resuspended at 1×10⁶/ml incomplete RPMI media 100 μl of cells were added to each well ofround-bottom 96 well plates. ODN solutions (100 μl) were added to wellsto give the selected range of final concentrations (1, 3, 6 μg/ml).Cells were cultured for 5 days and then pulsed with ³H-Thymidine (1μCi/well) for 18 h, before harvesting onto filter paper for measuringradioactivity. Results are reported as stimulation index (SI) withrespect to untreated media control.

Cytokine Assays

Freshly isolated PBMCs were resuspended at 10×10⁶/ml (2× finalconcentration) and 100 μl of cells were added to each well of a 96 wellflat-bottom plate containing an equal volume of ODN solution (2× finaldesired concentration). A range of concentrations (1, 3, 6 μg/ml) wastested for each ODN. Cells were incubated at 37° C. with 5% CO₂. After48 h, cell supernatants were collected from each well and frozen at −80°C. until assayed.

IFN-α, IP-10, IL-10 and IFN-_(.) levels in supernatants were measuredusing commercial ELISA Kits (R&D Systems, Minneapolis, Minn., USA;IP-10, Cat# DIP 100, IL-10, Cat# D1000, IFN-g Cat# DIF50 or PBLBiomedical, IFN-q Cat# 4110S). When measured ELISA values were below thedetection limit of the kit as specified by the manufacturer, a valueequal to the lowest detectable limit was entered into data tables.

Results

PBMCs isolated from blood collected from 15 chronically infected HCVsubjects and 10 normal healthy volunteers were incubated at 37° C. withdifferent classes of CpG (e.g., class A, B, C, soft C, semi-soft B andsemi-soft C), and cell supernatants were assessed for cytokine presence,indicative of cytokine secretion during the incubation period. Resultsof these experiments are presented below.

Induction of IFN-α Secretion by PBMC

When the three classes of CpG ODN were tested on PBMC from normalvolunteers, very high levels of IFN-α were produced by the A class (CpGSEQ ID NO. 1), moderately high levels by the C class (CpG SEQ ID NO. 4)and only low levels were induced by the B class (CpG SEQ ID NO. 2) (FIG.1). The main cellular source of IFN-α is pDC.

With the PBMC obtained from HCV chronic carriers, all three classes ofCpG could induce secretion of IFN-α. The levels with the B and C classeswere the same as those obtained with the normal PBMCs. In contrast, theA class induced only about 50% of the normal level (FIG. 1), suggestingthat the dysfunction of the HCV-infected pDC has some impact on theefficacy of A class CpG to induce IFN-α, but not the C class. Thus,either A or C class CpG could be used to treat HCV chronic carriers, butin some instances the C class may be preferred.

The number of pDC was determined by FACS analysis. A linear regressionwas performed against this compared to the amount of IFN-α secreted witheither the A and C class CpG ODN, and a reasonable correlation for thenormal subjects (e.g., R=0.43 and 0.58, respectively) was found. It wasfurther discovered that the correlation was slightly better for the Cclass ODNs. In contrast, no correlation was observed between number ofpDC and amount of IFN-α secreted for the HCV infected subjects (R=0.02and 0.08, respectively) (FIG. 3). The HCV-infected DC are neverthelesscapable of secreting IFN-α in response to the CpG ODN.

The effect of soft and semi-soft alterations of these ODS was alsoanalyzed. Soft molecules were synthesized that had a row ofphosphodiester bonds in the central region of the molecule. Semi-softmolecules were synthesized that had one or more individualphosphodiester bonds that are between the cytosine and guaninenucleotides of the CpG motifs. Both soft (FIG. 3) and semi-soft (FIG. 4)C class CpG ODN were capable of stimulating IFN-α secretion from normalor HCV PBMC in a manner similar to the original C class CpG ODN. Severalof the semi-soft C class CpG ODN were even more potent that the regularC class CpG SEQ ID NO. 4 (FIG. 4). This may be because the molecule isstill sufficient stable to have maximal immune stimulation and thephosphodiester in the middle of the CpG motif ?mass? increase itsactivity.

Induction of IFN-γ Secretion by PBMC

FIG. 5 compares the ability of different classes of CpG to induce thesecretion of Th1 cytokine, IFN-γ. Class A induced low levels of IFN-γwhile in comparison class B produced moderate amounts and class C CpGstimulated high concentrations of IFN-γ. Both HCV-infected and normalPBMCs displayed a similar Th1 response to all three classes of CpG.Similar results were obtained with semi-soft class C CpG ODN (FIG. 6).

Induction of IP-10 Secretion by PBMC

IP-10, a chemokine associated with production of type 1 and 2interferons, is also induced by CpG ODN. Highest levels are induced withA class, next highest with C class and lowest with B class CpG ODN.Regardless of the class of CpG ODN, similar levels of IP-10 were inducedwith PBMCs from normal subjects and HCV chronic carriers (FIG. 7).

B Cell Stimulation by CpG ODN

The effect of CpG on B cell stimulation was also investigated. As shownin FIGS. 8 and 9, CpG Class A was a poor stimulator of B cells for bothHCV-infected and normal populations. In contrast, classes B, C andsemi-soft C CpG strongly activated B cells. There were no differencesbetween PBMCs from normal and HCV-infected subjects.

IL-10 Secretion from PBMC after Stimulation with CpG ODN

The production of cytokine IL-10 following stimulation with CpG was alsoassessed and these results are shown in FIG. 10. For both HCV-infectedand normal subjects, all classes of CpG induced significant secretion ofIL-10, and there were no differences between PBMC from normal volunteersand those from HCV chronic carriers. Several cell types can produceIL-10 after incubation with CpG; however since B cells are the majorproducers of this cytokine, IL-10 production can be used as an indicatorof the level of B cell activation.

Effects of Intron A and Ribavirin

The in vitro effect of Ribavirin and exogenous IFN-α Intron A, alone orin combination with CpG was tested on HCV-infected cells. NeitherRibavirin nor Intron A, on their own or together, resulted in theinduction of IFN-α secretion by HCV-infected PBMCs (FIG. 11).

As has been discussed above, A and C class CpG ODN result in stronginduction of IFN-α secretion from pDC from normal and HCV-infectedsubjects. Furthermore, when CpG and Intron-A were used together, therewas a synergistic response for the majority (60%) of subjects (FIG. 12).

Discussion

CpG ODN are able to induce DC from patients chronically infected withHCV, to secrete IFN-α, with higher levels using Class A and C CpG andlower levels with Class B CpG ODN. The levels of secreted IFN-α arecomparable to those observed with cells from normal healthy volunteers.As well, IP-10 is induced from stimulated HCV PBMC, further indicating aTh1-type immune activation.

HCV antigen-specific immune responses are already present in personschronically infected with HCV. These are Th2-biased and thus cannotbring about clearance of the HCV-infected cells. Th1 type responseswould be required for viral clearance. Augmentation of systemic levelsof Th1cytokines, without additional antigen, allows persons chronicallyinfected with HCV to develop Th1-type HCV-specific immune responses thatare instrumental in viral clearance. All classes of CpG (A, B and C) arecapable of establishing Th1-type responses. These Th1-type responses areessential for long-term clearance of HCV chronic infection, yet they aredifficult to induce with exogenous IFN-α therapy, which has directanti-viral effects but not direct effects on the immune system. CpG ODNcan therefore be used in combination with exogenous IFN-α to treat HCVchronic carriers.

Alternatively, CpG ODN could also be used alone. Owing to induction ofcytokines such as IFN-α and IFN-γ, CpG ODN on its own has directanti-viral effects, in addition to the induction of Th1-typeHCV-specific immune responses. In some instances, the A and C classmolecules are preferred since they induce higher levels of IFNs.Depending on their characteristic sequence, CpG ODN can preferentiallystimulate pDC functions, maturation and type I IFN production (Krug, Aet al., Eur J Immunol, 2001; 31:2154-2163). Although according to theinvention two classes of CpG ODN were shown to be superior atstimulating IFN-α production, any CpG ODN, regardless of backbone or CpGsequence, could be used in the treatment of chronic HCV. The controlledrelease of different type I IFN isoforms by specific CpG ODN in vivo issuperior to the systemic administration of recombinant type I IFN thatis of a single subtype (e.g., Intron A is only IFN-α 2b). Soft andsemi-soft versions of CpG ODN are capable of stimulating similar levelsof IFN-α as their parent molecule. Soft or semi-soft versions of the CpGODN, especially the C class, would preferentially be used for chronictreatment of HCV, as they are more easily degraded and would thereforenot be expected to accumulate in the organs, specifically the liver,spleen and kidney.

At least 50% of the HCV subjects failed to respond to exogenous IFN-αtherapy, however CpG ODN (especially A and C class) were able to induceIFN-α secretion in vitro at levels comparable to normal healthyvolunteers in all subjects. CpG ODN could therefore be used to treatpatients who have failed to respond to exogenous IFN-α therapy, whetherthe IFN is pegylated or not, and whether the treatment also includesRibavirin or not. Classes of CpG ODN that induce high levels of IFN-αwould be preferred, and ever more preferred for long-term treatmentwould be the semi-soft versions.

Neither commercial Intron-A (IFN-α-2b) nor Ribavirin, alone or incombination, were capable of inducing IFN-α secretion from PBMCs fromnormal or HCV infected subjects in vitro. However when CpG ODN was usedin combination with Intron-A, a synergistic effect was observed forIFN-α secretion from PBMCs from HCV infected subjects. The C class ofODN were shown to have synergy with exogenous IFN-α; however treatmentwith any class of CpG ODN with commercial alpha-interferons would betherapeutically effective. As mentioned previously, due to theirrelative ease of degradation into non-stimulatory metabolites, semi-softversions of the CpG ODN could be used for chronic treatment without fearof accumulation in end-organs such as the kidney.

Ribavirin is purported to have Th1 effects, but in these studies it hadno immunostimulatory activity on human PBMCs. Even in combination withIntron A, Ribavirin did not enhance endogenous IFN-α production. Thus,replacing Ribavirin in combination therapy for HCV with a CpG ODN willincrease the proportion of sustained viral responses. When combined withCpG ODN, Ribavirin reduced the efficacy of CpG. CpG ODN should thereforebe given in combination with alpha-interferons in the absence ofRibavirin.

CpG ODN have been administered IM, SC and IV to human subjects and weredetermined to be well tolerated and safe (clinical study, in progress).Any effective route of administration would be acceptable such as SC,IM, IV, inhalation etc. however subcutaneous administration would be theroute of choice. CpG ODN were diluted in TE buffer and added to PBMCshowever, CpG ODN could also be formulated in delivery systems such asbioadhesive polymers (Sha et al., 1999), cochleates (Gould-Fogerite etal., 1994, 1996), dendrimers (Kukowska-Latallo et al.,1996, Qin et al,1998), enteric-coated capsules (Czerkinsky et al., 1987, Levine et al.,1987), emulsomes (Vancott et al., 1998, Lowell et al., 1997), ISCOMs(Mowat et al., 1993, Morein et al., 1999, Hu et al., 1998, Carlsson etal., 1991), liposomes (Childers et al., 1999, Michalek et al., 1989,1992), microspheres (Gupta et al., 1998, Maloy et al., 1994, Eldridge etal., 1989), nanospheres (Roy et al., 1999), polymer rings (Wyatt et al.,1998), proteosomes (Lowell et al., 1988, 1996) and virosomes (Gluck etal., 1992, Mengiardi et al., 1995, Cryz et al., 1998).

For treatment of HCV chronic carriers, CpG ODN could be administered ona repeated basis from once daily to once monthly, but preferably every3-10 days, and most preferably weekly, for a prolonged period. Thisperiod could be from one month to two years, but preferably 3 to 12months, and most preferably for 6 months. Thus the most optimal therapywould be given twice weekly or weekly for 6 months. It could also begiven more frequently during an inductive phase (daily or every otherday or twice weekly or weekly for the first 1-3 months), then lessfrequently for maintenance (weekly, or every other week, or monthly forseveral more months).

For combination therapy, CpG and alpha-interferons (pegylated or not)could potentially be (i) mixed together and given at the same time andby the same route (subcutaneous), (ii) given at the same time and sameroute but not mixed, (iii) given at the same time but by differentroutes (e.g., the alpha-interferon could be given SC and the CpG couldbe IV, IM, ID, orally or topically), (iv) given at different times andschedules with same or different routes, or (v) given consecutively. Inthis latter case, preferably the IFN-α would be given first in order toreduce viral load, then the CpG ODN would be given afterwards to induceand sustain Th1-type adaptive immunity for long term control.

REFERENCES

-   1. Choo, Q. L., G. Kuo, A. J. Weiner, L. R. Overby, D. W.    Bradley, M. Houghton. 1989. Isolation of a cDNA clone derived from a    blood-borne non-A, non-B viral hepatitis genome. Science. 244:    359-62.-   2. Choo, Q. L., K. H. Richman, J. H. Han, K. Berger, C. Lee, C.    Dong, C. Gallegos, D. Coit, R. Medina-Selby, P. J. Barr, et    al. 1991. Genetic organization and diversity of the hepatitis C    virus. Proc Natl Acad Sci USA. 88: 2451-5.-   3. van der Poel, C. L., H. T. Cuypers, H. W. Reesink. 1994.    Hepatitis C virus six years on. Lancet. 344: 1475-9.-   4. van der Poel, C. L. 1994. Hepatitis C virus. Epidemiology,    transmission and prevention. Curr Stud Hematol Blood Transfus:    137-63.-   5. Kiyosawa, K., T. Sodeyama, E. Tanaka, Y. Gibo, K. Yoshizawa, Y.    Nakano, S. Furuta, Y. Akahane, K. Nishioka, R. H. Purcell, et    al. 1990. Interrelationship of blood transfusion, non-A, non-B    hepatitis and hepatocellular carcinoma: analysis by detection of    antibody to hepatitis C virus. Hepatology. 12: 671-5.-   6. Alter, M. J., H. S. Margolis, K. Krawczynski, F. N. Judson, A.    Mares, W. J. Alexander, P. Y. Hu, J. K. Miller, M. A. Gerber, R. E.    Sampliner, et al. 1992. The natural history of community-acquired    hepatitis C in the United States. The Sentinel Counties Chronic    non-A, non-B Hepatitis Study Team. N Engl J Med. 327: 1899-905.-   7. Alter, M. J. 1994. Transmission of hepatitis C virus--route,    dose, and titer. N Engl J Med. 330: 784-6.-   8. Alter, M. J. 1994. Review of serologic testing for hepatitis C    virus infection and risk of posttransfusion hepatitis C. Arch Pathol    Lab Med. 118: 342-5.-   9. Alter, M. J., E. E. Mast. 1994. The epidemiology of viral    hepatitis in the United States. Gastroenterol Clin North Am. 23:    437-55.-   10. Weiner, A. J., H. M. Geysen, C. Christopherson, J. E.    Hall, T. J. Mason, G. Saracco, F. Bonino, K. Crawford, C. D.    Marion, K. A. Crawford, et al. 1992. Evidence for immune selection    of hepatitis C virus (HCV) putative envelope glycoprotein variants:    potential role in chronic HCV infections. Proc Natl Acad Sci USA.    89: 3468-72.-   11. Kato, N., Y. Ootsuyama, H. Sekiya, S. Ohkoshi, T. Nakazawa, M.    Hijikata, K. Shimotohno. 1994. Genetic drift in hypervariable region    1 of the viral genome in persistent hepatitis C virus infection. J    Virol. 68: 4776-84.-   12. Diepolder, H. M., R. Zachoval, R. M. Hoffmann, E. A.    Wierenga, T. Santantonio, M. C. Jung, D. Eichenlaub, G. R.    Pape. 1995. Possible mechanism involving T-lymphocyte response to    non-structural protein 3 in viral clearance in acute hepatitis C    virus infection. Lancet. 346: 1006-7.-   13. Missale, G., R. Bertoni, V. Lamonaca, A. Valli, M. Massari, C.    Mori, M. G. Rumi, M. Houghton, F. Fiaccadori, C. Ferrari. 1996.    Different clinical behaviors of acute hepatitis C virus infection    are associated with different vigor of the anti-viral cell-mediated    immune response. J Clin Invest. 98: 706-14.-   14. Tsai, S. L., Y. F. Liaw, M. H. Chen, C. Y. Huang, G. C.    Kuo. 1997. Detection of type 2-like T-helper cells in hepatitis C    virus infection: implications for hepatitis C virus chronicity.    Hepatology. 25: 449-58.-   15. Rehermann, B., K. M. Chang, J. G. McHutchison, R. Kokka, M.    Houghton, F. V. Chisari. 1996. Quantitative analysis of the    peripheral blood cytotoxic T lymphocyte response in patients with    chronic hepatitis C virus infection. Journal of Clinical    Investigation. 98: 1432-1440-   16. Erickson, A. L., M. Houghton, Q. L. Choo, A. J. Weiner, R.    Ralston, E. Muchmore, C. M. Walker. 1993. Hepatitis C virus-specific    CTL responses in the liver of chimpanzees with acute and chronic    hepatitis C. J Immunol. 151: 4189-99.-   17. Chen, M., M. Sallberg, A. Sonnerborg, O. Weiland, L.    Mattsson, L. Jin, A. Birkett, D. Peterson, D. R. Milich. 1999.    Limited humoral immunity in hepatitis C virus infection.    Gastroenterology. 116: 135-43.-   18. Nagler, A., L. L. Lanier, J. H. Phillips. 1988. The effects of    IL-4 on human natural killer cells. A potent regulator of IL-2    activation and proliferation. J Immunol. 141: 2349-51.-   19. Martinez, O. M., R. S. Gibbons, M. R. Garovoy, F. R.    Aronson. 1990. IL-4 inhibits IL-2 receptor expression and    IL-2-dependent proliferation of human T-cells. J Immunol. 144:    2211-5.-   20. Moore, K. W., O. G. A, R. de Waal Malefyt, P. Vieira, T. R.    Mosmann. 1993. Interleukin-10. Annu Rev Immunol. 11: 165-90.-   21. de Waal Malefyt, R., J. Haanen, H. Spits, M. G. Roncarolo, A. to    Velde, C. Figdor, K. Johnson, R. Kastelein, H. Yssel, J. E. de    Vries. 1991. Interleukin 10 (IL-10) and viral IL-10 strongly reduce    antigen-specific human T-cell proliferation by diminishing the    antigen-presenting capacity of monocytes via downregulation of class    II major histocompatibility complex expression. J Exp Med. 174:    915-24.-   22. Fiorentino, D. F., A. Zlotnik, P. Vieira, T. R. Mosmann, M.    Howard, K. W. Moore, O. G. A. 1991. IL-10 acts on the    antigen-presenting cell to inhibit cytokine production by Th1 cells.    J Immunol. 146: 3444-51.-   23. Schlaak, J. F., T. Pitz, H. F. Lohr, K. H. Meyer zum    Buschenfelde, G. Gerken. 1998. Interleukin 12 enhances deficient    HCV-antigen-induced Th1-type immune response of peripheral blood    mononuclear cells. J Med Virol. 56: 112-7.-   24. Cacciarelli, T. V., O. M. Martinez, R. G. Gish, J. C.    Villanueva, S. M. Krams. 1996. Immunoregulatory cytokines in chronic    hepatitis C virus infection: pre- and posttreatment with interferon    alfa. Hepatology. 24: 6-9.-   25. Kuzushita, N., N. Hayashi, K. Katayama, T. Kamada. 1995.    [Histological features and HLA-DNA types in HCV carriers with    persistently normal ALT levels]. Nippon Rinsho. 53: 576-81.-   26. Kanto, T., N. Hayashi, T. Takehara, T. Tatsumi, N. Kuzushita, A.    Ito, Y. Sasaki, A. Kasahara, M. Hori. 1999 Impaired allostimulatory    capacity of peripheral blood dendritic cells recovered from    hepatitis C virus-infected individuals. J Immunol. 162: 5584-91.-   27. Bain, C., A. Fatmi, F. Zoulim, J. P. Zarski, C. Trepo, G.    Inchauspe. 2001. Impaired allostimulatory function of dendritic    cells in chronic hepatitis C infection. Gastroenterology. 120:    512-24.-   28. Sansonno, D., C. Lotesoriere, V. Cornacchiulo, M. Fanelli, P.    Gatti, G. Iodice, V. Racanelli, F. Dammacco. 1998. Hepatitis C virus    infection involves CD34(+) hematopoietic progenitor cells in    hepatitis C virus chronic carriers. Blood. 92: 3328-37.-   29. Auffermann-Gretzinger, S., E. B. Keeffe, S. Levy. 2001. Impaired    dendritic cell maturation in patients with chronic, but not    resolved, hepatitis C virus infection. Blood. 97: 3171-6.-   30. Kruse, M., O. Rosorius, F. Kratzer, G. Stelz, C. Kuhnt, G.    Schuler, J. Hauber, A. Steinkasserer. 2000. Mature dendritic cells    infected with herpes simplex virus type 1 exhibit inhibited T-cell    stimulatory capacity. J Virol. 74: 7127-36.-   31. Fugier-Vivier, I., C. Servet-Delprat, P. Rivailler, M. C.    Rissoan, Y. J. Liu, C. Rabourdin-Combe. 1997. Measles virus    suppresses cell-mediated immunity by interfering with the survival    and functions of dendritic and T-cells. J Exp Med. 186: 813-23.-   32. Sarobe et.al. 2002, Journal of Virology 76:10, 5062-5070    Chisari, F. V., C. Ferrari. 1995. Hepatitis B virus    immunopathogenesis. Annu Rev Immunol. 13: 29-60-   33. Chisari, F. V. 1997. Cytotoxic T-cells and viral hepatitis.    Journal of Clinical Investigation. 99: 1472-1477-   34. Marianneau, P., A. M. Steffan, C. Royer, M. T. Drouet, D.    Jaeck, A. Kirn, V. Deubel. 1999. Infection of primary cultures of    human Kupffer cells by Dengue virus: no viral progeny synthesis, but    cytokine production is evident. J Virol. 73: 5201-6.-   35. Krieg, A. M., A. K. Yi, S. Matson, T. J. Waldschmidt, G. A.    Bishop, R. Teasdale, G. A. Koretzky, D. M. Klinman. 1995. CpG motifs    in bacterial DNA trigger direct B-cell activation. Nature. 374:    546-9-   36. Yi, A. K., P. Hornbeck, D. E. Lafrenz, A. M. Krieg. 1996. CpG    DNA rescue of murine B lymphoma cells from anti-IgM-induced growth    arrest and programmed cell death is associated with increased    expression of c-myc and bcl-xL. J Immunol. 157: 4918-4925-   37. Yi, A. K., J. H. Chace, J. S. Cowdery, A. M. Krieg. 1996.    IFN-gamma promotes IL-6 and IgM secretion in response to CpG motifs    in bacterial DNA and oligodeoxynucleotides. Journal of Immunology.    156: 558-64-   38. Klinman, D. M., A. K. Yi, S. L. Beaucage, J. Conover, A. M.    Krieg. 1996. CpG motifs present in bacteria DNA rapidly induce    lymphocytes to secrete interleukin 6, interleukin 12, and interferon    gamma. Proc Natl Acad Sci USA. 93: 2879-2883-   39. Klinman, D. M., D. Verthelyi, F. Takeshita, K. J. Ishii. 1999.    Immune recognition of foreign DNA: a cure for bioterrorism?    Immunity. 11: 123-9-   40. Halpern, M. D., R. J. Kurlander, D. S. Pisetsky. 1996. Bacterial    DNA induces murine interferon-gamma production by stimulation of    interleukin-12 and tumor necrosis factor-alpha. Cell Immunol. 167:    72-8-   41. Cowdery, J. S., J. H. Chace, A. K. Yi, A. M. Krieg. 1996.    Bacterial DNA induces NK cells to produce IFN-gamma in vivo and    increases the toxicity of lipopolysaccharides. J Immunol. 156:    4570-4575-   42. Schwartz, D. A., T. J. Quinn, P S Thorne, S. Sayeed, A. K.    Yi, A. M. Krieg. 1997. CpG motifs in bacterial DNA cause    inflammation in the lower respiratory tract. Journal of Clinical    Investigation. 100: 68-73-   43. Yamamoto, S., T. Yamamoto, T. Kataoka, E. Kuramoto, O. Yano, T.    Tokunaga. 1992. Unique palindromic sequences in synthetic    oligonucleotides are required to induce IFN [correction of INF] and    augment IFN-mediated natural killer activity. J Immunol. 148:    4072-6.-   44. Ballas, Z. K., W. L. Rasmussen, A. M. Krieg. 1996. Induction of    NK activity in murine and human cells by CpG motifs in    oligodeoxynucleotides and bacterial DNA. J Immunol. 157: 1840-1845-   45. Chace, J. H., N. A. Hooker, K. L. Mildenstein, A. M.    Krieg, J. S. Cowdery. 1997. Bacterial DNA-induced NK cell IFN-gamma    production is dependent on macrophage secretion of IL-12. Clinical    Immunology & Immunopathology. 84: 185-93-   46. Roman, M., E. Martin-Orozco, J. S. Goodman, M. D. Nguyen, Y.    Sato, A. Ronaghy, R. S. Kornbluth, D. D. Richman, D. A. Carson, E.    Raz. 1997. Immunostimulatory DNA sequences function as T    helper-1-promoting adjuvants [see comments]. Nat Med. 3: 849-54-   47. Krieg, A. M., S. Matson, K. Cheng, E. Fisher, G. A.    Koretzky, J. G. Koland. 1997. Identification of an    oligodeoxynucleotide sequence motif that specifically inhibits    phosphorylation by protein tyrosine kinases. Antisense and Nucleic    Acid Drug Development 7: 115-23-   48. Davis, H. L., R. Weeranta, T. J. Waldschmidt, L. Tygrett, J.    Schorr, A. M. Krieg. 1998. CpG DNA is a potent enhancer of specific    immunity in mice immunized with recombinant hepatitis B surface    antigen. Journal of Immunology. 160: 870-6-   49. Moldoveanu, Z., L. Love-Homan, W. Q. Huang, A. M. Krieg. 1998.    CpG DNA, a novel immune enhancer for systemic and mucosal    immunization with influenza virus. Vaccine. 16: 1216-24-   50. Chu, R. S., O. S. Targoni, A. M. Krieg, P. V. Lehmann, C. V.    Harding. 1997. CpG oligodeoxynucleotides act as adjuvants that    switch on T helper 1 (Th1) immunity. Journal of Experimental    Medicine. 186: 1623-31-   51. Lipford, G. B., M. Bauer, C. Blank, R. Reiter, H. Wagner, K.    Heeg. 1997. CpG-containing synthetic oligonucleotides promote B and    cytotoxic T-cell responses to protein antigen: a new class of    vaccine adjuvants. Eur J Immunol. 27: 2340-4-   52. Weiner, G. J., H. M. Liu, J. E. Wooldridge, C. E. Dahle, A. M.    Krieg. 1997. Immunostimulatory oligodeoxynucleotides containing the    CpG motif are effective as immune adjuvants in tumor antigen    immunization. Proceedings of the National Academy of Sciences of the    United States of America. 94: 10833-7-   53. Kline, J. N., T. J. Waldschmidt, T. R. Businga, J. E.    Lemish, J. V. Weinstock, P. S. Thorne, A. M. Krieg. 1998. Modulation    of airway inflammation by CpG oligodeoxynucleotides in a murine    model of asthma. J Immunol. 160: 2555-9-   54. Krieg, A. M. 2001. Now I know my CpGs. Trends Microbiol. 9:    249-52.-   55. Krieg, A. M., L. Love-Homan, A. K. Yi, J. T. Harty. 1998. CpG    DNA induces sustained IL-12 expression in vivo and resistance to    Listeria monocytogenes challenge. J Immunol. 161: 2428-34-   56. Wallcer, P. S., T. Scharton-Kersten, A. M. Krieg, L.    Love-Homan, E. D. Rowton, M. C. Udey, J. C. Vogel. 1999.    Immunostimulatory oligodeoxynucleotides promote protective immunity    and provide systemic therapy for leishmaniasis via IL-12- and    IFN-gamma-dependent mechanisms. Proc Natl Acad Sci USA. 96: 6970-5-   57. Gramzinski, R. A., D. L. Doolan, M. Sedegah, H. L. Davis, A. M.    Krieg, S. L. Hoffman. 2001. Interleukin-12- and gamma    interferon-dependent protection against malaria conferred by CpG    oligodeoxynucleotide in mice. Infect Immun. 69: 1643-9.-   58. Roffi, L., G. C. Mels, G. Antonelli, G. Bellati, F.    Panizzuti, A. Piperno, M. Pozzi, D. Ravizza, G. Angeli, F. Dianzani,    et al. 1995. Breakthrough during recombinant interferon alfa therapy    in patients with chronic hepatitis C virus infection: prevalence,    etiology, and management. Hepatology. 21: 645-9.-   59. Imai, Y., S. Kawata, S. Tamura, I. Yabuuchi, S. Noda, M.    Inada, Y. Maeda, Y. Shirai, T. Fukuzaki, I. Kaji, H. Ishikawa, Y.    Matsuda, M. Nishikawa, K. Seki, Y. Matsuzawa. 1998. Relation of    interferon therapy and hepatocellular carcinoma in patients with    chronic hepatitis C. Osaka Hepatocellular Carcinoma Prevention Study    Group. Ann Intern Med. 129: 94-9.-   60. Davis H L C C, Morris M L, Efler S M, Cameron D W,    Heathcote J. 2000. CpG ODN is safe and highly effective in humans as    adjuvant to HBV vaccine: Preliminary results of Phase I trial with    CpG ODN SEQ ID NO. 2. Presented at The Third Annual Conference on    Vaccine Research. S25: 47.

EQUIVALENTS

It will be understood that various modifications may be made to theembodiments disclosed herein. Therefore, the above description shouldnot be construed as limiting, but merely as exemplifications ofpreferred embodiments. Those skilled in the art will envision othermodifications within the scope of the claims appended hereto.

All references, patents and patent applications disclosed herein areincorporated by reference in their entirety.

1. A method of treating a subject having an HCV infection that was notsuccessfully treated using a previous non-CpG therapy comprisingadministering to a subject in need of such treatment a CpGimmunostimulatory nucleic acid in an amount effective to treat theinfection.
 2. The method of claim 1, wherein the non-CpG therapyincludes interferon-alpha.
 3. The method of claim 2, wherein theinterferon-alpha is interferon-alpha-2b, interferon-alpha-2a orconsensus interferon-alpha.
 4. The method of claim 2, wherein thenon-CpG therapy includes interferon-alpha and Ribavirin.
 5. The methodof claim 2, wherein the non-CpG therapy includes pegylatedinterferon-alpha and Ribavirin.
 6. The method of claim 1, wherein theCpG immunostimulatory nucleic acid is an A class CpG immunostimulatorynucleic acid.
 7. The method of claim 1, wherein the CpGimmunostimulatory nucleic acid is a B class CpG immunostimulatorynucleic acid
 8. The method of claim 1, wherein the CpG immunostimulatorynucleic acid is a C class CpG immunostimulatory nucleic acid.
 9. Themethod of claim 1, further comprising the step of administeringinterferon-alpha to the subject.
 10. The method of claim 9, wherein theinterferon-alpha is interferon-alpha-2b, interferon-alpha-2a orconsensus interferon alpha.
 11. The method of claim 9, wherein theinterferon-alpha is administered substantially simultaneously with theCpG immunostimulatory nucleic acid.
 12. The method of claim 1, whereinthe CpG immunostimulatory nucleic acid comprises a backbonemodification.
 13. The method of claim 12, wherein the backbonemodification is a phosphorothioate backbone modification.
 14. The methodof claim 1, wherein the CpG immunostimulatory nucleic acid comprises asemi-soft backbone.
 15. A method of treating a subject having an HCVinfection and likely to be non-responsive to a non-CpG therapycomprising administering to a subject in need of such treatment a CpGimmunostimulatory nucleic acid in an amount effective to treat theinfection.
 16. The method of claim 15, further comprising identifying asubject likely to be non-responsive to a non-CpG therapy.
 17. The methodof claim 16, wherein the subject is identified as likely to benon-responsive based on an assay of interferon-alpha produced perdendritic cell.
 18. The method of claim 16, wherein the subject isidentified as likely to be non-responsive based on HCV genotype.
 19. Themethod of claim 15, wherein the non-CpG therapy includesinterferon-alpha.
 20. The method of claim 15, wherein the non-CpGtherapy includes interferon-alpha and Ribavirin.
 21. The method of claim20, further comprising administering to the subject an anti-viral agent.22. The method of claim 21, wherein the anti-viral agent isinterferon-alpha.
 23. The method of claim 22, wherein theinterferon-alpha is interferon-alpha-2b, interferon-alpha-2a orconsensus interferon alpha.
 24. The method of claim 21, whereininterferon-alpha administered in a sub-therapeutic amount.
 25. Themethod of claim 15, wherein the CpG immunostimulatory nucleic acid is aC class CpG immunostimulatory nucleic acid.
 26. The method of claim 15,wherein the CpG immunostimulatory nucleic acid comprises a semi-softbackbone.
 27. A method for screening CpG immunostimulatory nucleic acidsuseful in the treatment of chronic hepatitis C viral infectioncomprising contacting peripheral blood mononuclear cells from a subjecthaving a chronic hepatitis C viral infection, with a CpGimmunostimulatory nucleic acid, and measuring a test response of theblood mononuclear cells after exposure, wherein the subject was notsuccessfully treated using a previous therapy.
 28. The method of claim27, wherein the test response is selected from the group consisting of Bcell stimulation, secretion of IL-6, secretion of IL-10, secretion ofIL-12, secretion of interferon-gamma, secretion of type 1 interferons(alpha +beta), secretion of IP-10, NK activity, expression of CD80,expression of CD 86, expression of CD83, and upregulation of class IIMHC expression.
 29. The method of claim 27, wherein the peripheral bloodmononuclear cells comprise dendritic cells.
 30. The method of claim 29,wherein the dendritic cells comprise plasmacytoid dendritic cells. 31.The method of claim 29, wherein the test response is selected from thegroup consisting of secretion of IL-12, secretion of type 1 interferons,expression of CD80, expression of CD 86, expression of CD83, andupregulation of class II MHC expression.
 32. The method of claim 29,wherein the contacting occurs in vitro.
 33. The method of claim 32,wherein the peripheral blood mononuclear cells are cultured.
 34. Themethod of claim 33, wherein the CpG immunostimulatory nucleic acid isadded to the cultured peripheral blood mononuclear cells.
 35. The methodof claim 29, wherein the previous therapy is a non-CpG therapy.
 36. Themethod of claim 29, wherein the previous therapy is therapy with a CpGnucleic acid of a different sequence or class.
 37. The method of claim29, further comprising screening the CpG immunostimulatory nucleic acidfor the ability to stimulate a control response from peripheral bloodmononuclear cells from a normal subject.
 38. The method of claim 29,further comprising contacting peripheral blood mononuclear cells tointerferon-alpha substantially simultaneously with the CpGimmunostimulatory nucleic acid.
 39. The method of claim 29, wherein theCpG immunostimulatory nucleic acid is a C class CpG immunostimulatorynucleic acid.
 40. A method for identifying a subject having an HCVinfection and likely to be non-responsive to a non-CpG therapycomprising exposing peripheral blood mononuclear cells harvested from asubject having a hepatitis C viral infection to a CpG immunostimulatorynucleic acid, measuring interferon-alpha produced from the cells, anddetermining an amount of interferon-alpha produced per dendritic cell,wherein an amount that is below 1.0 pg/ml is indicative of a subjectthat is likely to be non-responsive to a non-CpG therapy.
 41. The methodof claim 40, wherein an amount that is below 0.5 pg/ml is indicative ofa subject that is likely to be non-responsive to a non-CpG therapy. 42.The method of claim 40, wherein the non-CpG therapy comprisesinterferon-alpha.
 43. The method of claim 42, wherein the non-CpGtherapy comprises Ribavirin.
 44. The method of claim 42, wherein theIFN-alpha is pegylated interferon-alpha.
 45. The method of claim 40,wherein the CpG immunostimulatory nucleic acid is an A class or a Cclass CpG immunostimulatory nucleic acid.
 46. The method of claim 40,wherein the peripheral blood mononuclear cells are further exposed to ananti-viral agent together with a CpG immunostimulatory nucleic acid. 47.The method of claim 46, wherein the anti-viral agent isinterferon-alpha.
 48. The method of claim 47, wherein interferon-alphais interferon-alpha-2b, interferon-alpha-2a or consensus interferonalpha.
 49. The method of claim 40, wherein the peripheral bloodmononuclear cells comprise dendritic cells.
 50. The method of claim 49,wherein the dendritic cells comprise plasmacytoid dendritic cells. 51.The method of claim 40, wherein the hepatitis C viral infection is anacute hepatitis C viral infection.
 52. The method of claim 40, furthercomprising determining a genotype of the HCV.
 53. A method of treating asubject having a hepatitis C viral infection comprising administering toa subject identified according to the method of claim 40 a CpGimmunostimulatory nucleic acid molecule in an amount effective to treatthe infection.
 54. The method of claim 53, further comprisingadministering to the subject interferon-alpha.
 55. The method of claim54, wherein the interferon-alpha is interferon-alpha-2b,interferon-alpha-2a or consensus interferon-alpha.
 56. The method ofclaim 53, wherein the CpG immunostimulatory nucleic acid is an A classCpG immunostimulatory nucleic acid.
 57. The method of claim 53, whereinthe CpG immunostimulatory nucleic acid is a B class CpGimmunostimulatory nucleic acid.
 58. The method of claim 53, wherein theCpG immunostimulatory nucleic acid is a C class CpG immunostimulatorynucleic acid.
 59. The method of claim 53, wherein the CpGimmunostimulatory nucleic acid comprises a backbone modification. 60.The method of claim 59 wherein the backbone modification is aphosphorothioate backbone modification.
 61. The method of claim 53,wherein the CpG immunostimulatory nucleic acid comprises a semi-softbackbone.
 62. The method of claim 53, wherein the hepatitis C viralinfection is a chronic hepatitis C viral infection.
 63. The method ofclaim 53, wherein the hepatitis C viral infection is an acute hepatitisC viral infection.
 64. A method of treating a subject having an HCVinfection that was not successfully treated using a previous non-CpGtherapy comprising administering to a subject in need of such treatmenta C class CpG immunostimulatory nucleic acid having a semi-soft backbonein an amount effective to treat the infection.
 65. A method of treatinga subject having an HCV infection and likely to be non-responsive to anon-CpG therapy comprising administering to a subject in need of suchtreatment a C class CpG immunostimulatory nucleic acid having asemi-soft backbone in an amount effective to treat the infection.
 66. Amethod for identifying a subject having an HCV infection and likely tobe non-responsive to a non-CpG therapy comprising exposing peripheralblood mononuclear cells harvested from a subject having a hepatitis Cviral infection to a A class or a C class CpG immunostimulatory nucleicacid, measuring interferon-alpha produced from the cells, anddetermining an amount of interferon-alpha produced per dendritic cell,wherein an amount that is below 1.0 pg/ml is indicative of a subjectthat is likely to be non-responsive to a non-CpG therapy.
 67. A methodof treating a subject having an HCV infection that was not successfullytreated using a previous non-CpG therapy comprising contactingperipheral blood mononuclear cells from a subject in need of suchtreatment, with a CpG immunostimulatory nucleic acid in an amounteffective to stimulate an immune response, and re-infusing the cellsinto the subject.
 68. The method of claim 67, wherein the peripheralblood mononuclear cells comprise dendritic cells.
 69. The method ofclaim 68, wherein the dendritic cells comprise plasmacytoid dendriticcells.
 70. The method of claim 67, wherein the CpG immunostimulatorynucleic acid is a C class immunostimulatory nucleic acid.
 71. The methodof claim70, wherein the C class immunostimulatory nucleic acid has asemi-soft backbone.